Centre for Research in Neuroscience, McGill University, The Montreal General Hospital Research Institute, Montréal, Qúebec, Canada H3G 1A4
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Abstract |
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To investigate the role of the neurofilament
heavy (NF-H) subunit in neuronal function, we generated mice bearing a targeted disruption of the gene
coding for the NF-H subunit. Surprisingly, the lack of
NF-H subunits had little effect on axonal calibers and
electron microscopy revealed no significant changes in
the number and packing density of neurofilaments
made up of only the neurofilament light (NF-L) and
neurofilament medium (NF-M) subunits. However, our
analysis of NF-H knockout mice revealed an ~2.4-fold increase of microtubule density in their large ventral
root axons. This finding was further corroborated by a
corresponding increase in the ratio of assembled tubulin to NF-L protein in insoluble cytoskeletal preparations from the sciatic nerve. Axonal transport studies
carried out by the injection of [35S]methionine into spinal cord revealed an increased transport velocity of
newly synthesized NF-L and NF-M proteins in motor axons of NF-H knockout mice. When treated with ,
'-iminodipropionitrile (IDPN), a neurotoxin that segregates microtubules and retards neurofilament transport, mice heterozygous or homozygous for the NF-H
null mutation did not develop neurofilamentous swellings in motor neurons, unlike normal mouse littermates. These results indicate that the NF-H subunit is a
key mediator of IDPN-induced axonopathy.
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Introduction |
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NEUROFILAMENTS are made up by the copolymerization of three intermediate filament proteins,
neurofilament light (NF-L)1 (61 kD), neurofilament medium (NF-M) (90 kD), and neurofilament heavy
(NF-H) (110 kD) (Hoffman and Lasek, 1975; Liem et al.,
1978
). In the mouse, neurofilaments are obligate heteropolymers requiring NF-L with either NF-M or NF-H for
polymer formation (Ching and Liem, 1993
; Lee et al.,
1993
). A remarkable feature of the NF-H protein is its
long COOH-terminal tail domain that forms side-arm projections at the periphery of the filament. The tail NF-H domain is rich in charged amino acids and has multiple repeats Lys-Ser-Pro (KSP) that account for its unusual high
content of phosphoserine residues (Julien and Mushynski,
1982
; Carden et al., 1985
; Julien et al., 1988
; Lees et al.,
1988
). The presence of charged amino acids in NF-M and
NF-H led to the suggestion that repulsive forces between
neurofilaments would affect neurofilament packing density and axonal caliber. Evidence for this notion has come
from studies on the dysmyelinating mutant Trembler
mouse (deWaegh et al., 1992
) and on hypomyelinating
transgenic mice expressing in Schwann cells either a diphtheria toxin A or SV-40 large T antigen in which reduced
levels of NF-H phosphorylation resulted in decreases in axon caliber (Cole et al., 1994
). In addition, there is evidence that the NF-H protein may act as a modulator of axonal transport. The appearance of NF-H protein during
postnatal development coincides with slowing of axonal
transport (Willard and Simon, 1983
), whereas overexpression of either human or mouse NF-H proteins in transgenic mice cause an impairment of neurofilament transport (Collard et al., 1995
; Marszalek et al., 1996
). The
precise mechanism by which NF-H overexpression can reduce neurofilament transport remains unknown.
The abnormal accumulations of neurofilaments in distinct regions of the neuron occur in a variety of disorders
including amyotrophic lateral sclerosis (ALS) (Carpenter,
1968; Hirano et al., 1984
; Chou, 1992
), an inherited giant
axonal neuropathy in children (Carpenter et al., 1974
), and
toxic neuronopathies induced by, 2,5-hexanedione (Graham et al., 1984
), acrylamide (Asbury and Johnson, 1978
),
aluminium (Troncoso, 1992), and
,
'-iminodipropionitrile (IDPN) (Griffin et al., 1978
). The pathology in the
case of IDPN is of particular interest because this agent induces abnormal neurofilamentous accumulations in proximal axons similar to those found in ALS (Griffin et al.,
1978
). The molecular mechanism underlying IDPN-mediated pathology remains unknown but it is well established that IDPN intoxication causes segregation of neurofilaments from microtubules. Increases in immunoreactivity
of NF-H phosphorylation-related epitopes have been reported after IDPN treatment (Watson et al., 1989
). However, during the early phase of IDPN intoxication a transient dephosphorylation of NF-H precedes an increase in
the proportion of cold-soluble tubulin and the formation
neurofilament accumulations. These early changes suggested a role for the NF-H protein in modulating neurofilament-microtubule interactions and perhaps microtubule
stabilization (Tashiro et al., 1994
).
To further investigate the role of NF-H in neuronal function and in toxin-induced axonopathy, we generated mice bearing a targeted disruption of the NF-H gene using the technique of homologous recombination in embryonic stem cells. We report here that the absence of NF-H had no significant effect on neurofilament formation but it resulted in higher microtubule density in large motor axons and it increased velocity of neurofilament transport. Moreover, IDPN treatment of mice homozygous or heterozygous for the NF-H null mutation revealed that the NF-H protein is a key mediator of axonopathy induced by this agent.
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Materials and Methods |
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Targeting Vector
The mouse genomic NF-H DNA was isolated from a mouse phage genomic library as described previously (Julien et al., 1988). The targeting
vector was constructed by insertion of a blunt-ended 1.1-kb XhoI and
BamHI fragment of pMC1neoPoly A (Stratagene, La Jolla, CA) into the
second XmaI site of the NF-H gene as shown in the Fig. 1 A. The final targeting fragment was excised out by BamHI digestion and then purified by
b-Agarase (GIBCO BRL, Gaithersburg, MD).
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Cell Culture, Embryo Microinjection, and Animal Breeding
The culture of R1 embryonic stem (ES) cells (a gift from J. Roder, Mount
Sinai Research Institute, Toronto, Canada) and transfection were carried
out as previously described (Mansour et al., 1988; Ramirez-Solis et al.,
1993
). ES cells (107) were transfected with 25 mg of purified targeting DNA
using the BRL Cell Porator at 330 mF 230 V in DME at room temperature. The cells were treated with 180 mg/ml of G418 at 24 h after electroporation for 3 d, and then the concentration of G418 was increased to 360 mg/ml. After 7-8 d under G418 selection, colonies were picked up, trypsinized, and
then plated onto 96-well plates with SNL feeder layer (feeder layer and LIF
1,000 units/ml were used all time in the ES cell culture). Cells were
trypsinized 4 d later and 30% of the cells were plated for DNA extraction
onto gelatin-coated 96-well plates while 70% of them were frozen in duplicate plates. DNA was extracted after 5 d of culture and digested with EcoRI in the plates (Ramirez-Solis et al., 1993
). The ES cell clones that yielded an
expected 2.8-kb recombinant band on Southern blot (see Fig. 1) were expanded and reprobed by Neo probe to exclude the clones with random insertions. Three ES cell clones were injected into C57BL/6 blastocysts as described before (Mansour et al., 1988
). Germline transmission was obtained
by mating male chimeras with C57BL/6 females. The use of animals and all
surgical procedures described in this article were carried out according to
The Guide to the Care and Use of Experimental Animals of the Canadian
Council on Animal Care (see http://www.pharma.mcgill.ca/grant.htm).
RNA Analysis
Mice were killed by pentobarbital overdose and tissue samples were immediately frozen after dissection in liquid nitrogen and stored at 80°C.
Total RNA was isolated by homogenization in guanidinium thiocyanate
according to the method of Chomczynski and Sacchi (1987)
. The RNA
samples (20 mg) were fractionated on a 1% agarose-formaldehyde gel before blotting on a Hybond N+ membrane (Amersham Corp., Arlington
Heights, IL). The DNA probes were labeled with [
-32P]dATP by random
priming. Hybridizations were performed for 16 h at 42°C in 50% formamide, 1 M NaCl, 50 mM Tris, pH 7.5, 0.2% Ficoll, 0.2% polyvinyl pyrrolidone, 0.1% sodium pyrophosphate, 1% SDS, and 100 mg/ml sonicated
and denatured salmon sperm DNA. Blots were washed in 2× SSC-0.1%
SDS at room temperature for 10 min, in 2× SSC-0.1% SDS at 65°C twice
for 10 min, and in 0.5× SSC-0.1% SDS at room temperature once for 15 min. Blots were exposed to film (X-OMat AR; Eastman Kodak, Rochester, NY) with intensifying screens for 18 h at
80°C.
Western Blot Analysis
The mice were killed by intra-peritoneal injection of overdose pentobarbital and the nervous tissue samples were frozen in liquid nitrogen and
stored at 80°C. Total protein extracts were obtained by homogenization
of samples in SDS-urea buffer (0.5% SDS, 8 M urea in 7.4 phosphate
buffer). The supernatant was collected after centrifugation at 10,000 g for
20 min. The protein concentration was estimated by the Bradford procedure (Bio-Rad Laboratories, Hercules, CA). The proteins (10 mg) were
fractionated on a 7.5% SDS-PAGE and either visualized by Coomassie
blue staining or blotted onto a nitrocellulose membrane for Western blot
analysis. The mAbs against NF-L and NF-H were purchased from Amersham Corp. and Sternberger Inc. (Lutherville, MD), respectively. The
mAbs against tau, tubulin, and actin were purchased from Boehringer Mannheim Corp. (Indianapolis, IN). The mAb against acetylated tubulin
was purchased from Sigma Chemical Co. (St. Louis, MO). The Western
blot results were revealed by Renaissance® (a Western blot chemiluminescence kit from Dupont-NENTM, Boston, MA).
IDPN Intoxication
Five sets of 2-3-mo-old NF-H +/+, NF-H +/, and NF-H
/
littermates were intoxicated with two doses (first day and fifth day) of IDPN
intraperitoneally (1.5 g/kg body weight in 50% saline solution; Aldrich
Chemical Co., Milwaukee, WI) and maintained on 0.02% IDPN in drinking water. IDPN-treated mice were analyzed at 0, 7, and 15 d after intoxication.
Histological Methods
Mice were killed by overdose of pentobarbital, perfused with 0.9% NaCl, and then with fixative (2.5% glutaraldehyde, 0.5% PFA in 0.1 M sodium phosphate buffer, pH 7.4). Tissue samples were immersed in fixative for 2 h, rinsed in phosphate buffer, and then postfixed in 1% phosphate buffered osmium tetroxide. After three washes with phosphate buffer, each sample was dehydrated in a graded series of ethanol and embedded in Epon. The thin sections were stained with Toluidine blue and examined under a Polyvar microscope. The counting of axons and evaluation of myelin thickness in the L5 ventral root were carried out with the Image-1 software from Universal Imaging Corporation (West Chester, PA). The ultrathin sections were stained with lead citrate and examined with a Philips CM10 electron microscope (Philips Electron Optics, Mahwah, NJ).
Ultrastructural Morphometry Analysis
Ultrathin cross sections (100 nm) of L5 ventral root axons (>5 mm in
diam) at the dorsal root ganglia (DRG) region were photographed at a
magnification of 11,500× and enlarged to a final magnification of 81,650×
by printing. Positions of 10-nm filaments were marked by puncturing the
print with a needle, and then captured by a CCD camera and digitized using Imagine I. Digitized x-y coordinates were then computed by an IBM
program written in C++ programming language. Nearest neighbor distance was calculated for each filament. Neurofilament packing density was
calculated using the tile-counting procedure as previously reported (Hsieh
et al., 1994) with the following modifications. Square tiles of 100 nm × 100 nm were used, and tiles that did not contain any end-on neurofilaments were eliminated by the program.
The density and distribution of microtubules vary substantially according to the type of neurons, axonal caliber size, and myelination status of
the axon (Hsieh et al., 1994). Analysis of microtubules was carried out as
follows. Ultrathin cross sections (100 nm) of L5 ventral root axons of 5.0-
5.5 mm in diameter at the level of DRG region having a clear myelin
sheath were photographed at a magnification of 3,100× and enlarged another 8 times by printing. The position of 25-nm microtubules were taken
in the same way as described above for neurofilament calculation. Axon
area for the density calculation did not include myelin sheath. All calculations were done by a computer program with a graphic display written specifically for this application.
Taxol-stabilized Microtubule Preparations
Sciatic nerves between DRG and obturator tendon were dissected out
from newly killed mice and homogenized in a 2-ml glass-to-glass tissue
grinder in 1 ml of taxol/Triton X-100 PHEM extraction medium (60 mM
Pipes, 25 mM Hepes, 10 mM EGTA, 2 mM MgCl, 0.1% DMSO 10 µg/ml
leupeptin, and 0.6 mM PMSF) (Black et al., 1986). The homogenates were
incubated at room temperature for 30 min to solubilize unassembled tubulin and then centrifuged for 45 min in a Beckman TLA 45 rotor at 30,000 rpm. The pellets were dissolved in 100 µl of 1% SDS-Tris buffer (50 mM
Tris, pH 7.4) and the protein content was estimated by the Bio-Rad Dc
Protein Assay kit (Bio-Rad Modified Lowry method). The proteins (10 µg) were fractionated on a 7.5% SDS-PAGE and either visualized by
Coomassie blue staining or blotted on a nitrocellulose membrane for
Western blot analysis. Densitometric analysis was carried out by the Gel-ProTM Analyzer (version 2.0) from Meia cybernetics (Silver Spring, MD).
Metabolic Labeling
Proteins carried by slow axonal transport in sciatic motor axons were metabolically labeled and fractionated mainly as previously reported (Kirkpatrick and Brady, 1994). Aliquots of 0.5 mCi of [35S]methionine (DuPont-NEN) in 1-ml vol were injected into the anterior horn area of L2-L4
spinal cord twice on each side, 1-mm deep from the dorsal surface and 1 mm from the middle groove at a rate of 0.05 ml/min. 30 d after labeling,
the sciatic nerves with ventral roots (L3 to L5) were dissected out from
both sides and cut into 3-mm consecutive segments. Proteins were fractionated by SDS-PAGE and radiolabeled proteins detected by fluorography.
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Results |
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Targeted Disruption of NF-H Gene in Mice
A genomic fragment of NF-H was isolated from a mouse
library using a mouse cDNA probe (Julien et al., 1986).
The mouse NF-H gene contains four exons separated by
three introns (Julien et al., 1988
). A targeting vector of ~8
kb was constructed by inserting a TK/Neo cassette into the
XmaI site of exon I as shown in Fig. 1 A. The DNA fragment was transfected by electroporation into R1 ES cells
and neomycin resistance colonies were selected (Nagy et
al., 1993
). A total of 288 G418-resistant clones were picked up. Three ES cell clones out of eleven selected for homologous recombination event were microinjected into mouse
blastocysts to generate chimeric mice. The majority of chimeras derived from this ES cell line were males and they
were able to transmit agouti coat color to their progeny
when mated to C57BL/6 females. About half of agouti
progeny (F1) were heterozygous for the NF-H null mutation. The genotype of the mice was determined by Southern blot analysis of tail DNA. A Mendelian transmission
of the NF-H disrupted gene was obtained by the breeding
of heterozygous NF-H +/
F1 males and females. Thus,
the NF-H null mutation in mice does not cause lethality
during embryonic development or after birth. The homozygous NF-H knock-out mice were fertile and appeared phenotypically normal.
Levels of NF-L and NF-M in the Absence of NF-H
Northern blot analysis was carried out with 10 µg of total
brain mRNA of adult NF-L +/+, +/, and
/
mice (Fig.
1 B). No NF-H mRNA was detected in NF-H knockout
mice while ~50% decrease in NF-H mRNA was detected
in NF-H heterozygous +/
mice. The depletion of NF-H
mRNA did not affect the level of expression of either NF-M
or NF-L mRNAs. Coomassie blue-stained gels of Triton
X-100-insoluble extracts at 4°C from the spinal cord and
sciatic nerve showed absence of NF-H protein in NF-H
/
mice (Fig. 1 C). This was further confirmed by immunoblotting (Fig. 1 D). No band corresponding to NF-H was
detected with SMI-31 and SMI-32 antibodies (Sternberger Inc.) that recognizes the hyperphosphorylated and hypophosphorylated forms of NF-H, respectively. The lack of
NF-H protein did not affect the levels of NF-L protein but
it resulted in a modest increase of ~20% in NF-M levels
(Fig. 1 C). Thus, unlike the situation with NF-L knockout
mice where dramatic decreases in the levels of both NF-M
and NF-H proteins were observed (Zhu et al., 1997
), the
absence of NF-H did not cause dramatic changes on the
stability of the other two neurofilament subunits, NF-M
and NF-L. However, it is noteworthy that the loss of NF-H
protein increased dramatically the signal of NF-M cross-reactivity with the SMI-31 antibody (Fig. 1 D). This is
likely due to a compensatory increase in the phosphorylation state of NF-M that contains, like NF-H, multiple Lys-Ser-Pro phosphorylation sites (Levy et al., 1987
; Myers et
al., 1987
).
No Substantial Changes in Density of Neurofilaments and in Calibers of Axons
Since there are some lines of evidence suggesting a role for
NF-H protein in neurofilament spacing and axonal caliber
(deWaegh et al., 1992; Hsieh et al., 1994
; Nixon et al.,
1994
), light and electron microscopy was carried out on L5
ventral roots of adult NF-H +/+, NF-H +/
, and NF-H
/
mice. Surprisingly, the NF-H depletion caused only
modest changes in the caliber of myelinated axons (Figs. 2
and 3 A). Like NF-H +/+ mice, the NF-H
/
mice yielded a bimodal distribution of axonal calibers representing the small and large myelinated axons (Fig. 3 A). In
the NF-H
/
mice, the caliber of large myelinated axons
was only slightly smaller (~10%) than those of normal
mice. To examine whether interfilament spacing was affected in large ventral root axons lacking NF-H, we recorded the positions of each filament in individual axons and calculated nearest neighbor distances for each neurofilament. No significant changes in the number of assembled neurofilaments were observed in NF-H +/
and
NF-H
/
mice (Fig. 3 B). The NF-H subunit was clearly
dispensable for the formation of 10-nm filaments and its
absence caused no substantial changes in the average
nearest neighbor distance, neurofilament packing density
and axon caliber (Figs. 3 and 4).
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Increased Density of Microtubules in Ventral Root Axons
The most remarkable change in the axonal cytoskeleton of
ventral root axons lacking NF-H was a ~2.4-fold increase
in microtubule content (Fig. 3 D). Considering that the
density and distribution of microtubules may vary according to the type of neurons, axonal caliber size, and myelination status of the axon (Hsieh et al., 1994), our analysis
was carried out on large myelinated axons (~20 mm2 ± 2.5 axoplasmic area) of the L5 ventral root, a well-defined population of axons. We counted the number of microtubules on electron micrographs of individual transverse sections of axons magnified 25,000×. Our study involved four
pairs of NF-H +/+ and NF-H
/
littermates with two
sets derived from three distinct ES cell clones and, for
each animal, 7-24 axons were analyzed. The number of
microtubules per axonal area (20 mm2) was of 193 ± 86 (n = 50) in myelinated axons from control littermates and
of 463 ± 94 (n = 58) in the NF-H knockout mice.
Taxol-stabilized cytoskeletal preparations from the sciatic nerve were analyzed to further confirm whether the
levels of assembled tubulin were increased. Sciatic nerves
between DRG and obturator tendon were dissected and
homogenized in taxol/Triton X-100 extraction buffer as
described previously (Black et al., 1986). After 30 min at
room temperature, the homogenates were centrifuged at
45,000 g for 45 min. The pellets were dissolved in 1%
SDS-Tris buffer (50 mM Tris, pH 7.4) and the proteins
fractionated on SDS-PAGE. As shown on the Coomassie
blue-stained gel of these cytoskeletal preparations and
corresponding histograms in Fig. 5, the levels of assembled
tubulin was increased of ~2.5-fold in samples from the
NF-H knockout mice as compared with normal mice. The
rise in assembled microtubules in axons lacking NF-H was
not due to an increase of total tubulin levels in the sciatic
nerve (Fig. 1 D) implying that the absence of NF-H contributed to increase the stability of microtubules. We examined by immunoblotting using the TAU-1 antibody
whether the levels of tau, a microtubule-associated protein
(MAP) that suppresses microtubule dynamics, were altered in the sciatic nerve of NF-H
/
mice. No changes
in levels of tau were evident in samples from total nerve
extracts (data not shown). However, the TAU-1 antibody
detected elevated levels of low molecular weight tau in cytoskeletal preparations from the sciatic nerve of NF-H
/
mice (Fig. 6). From these results, it is possible that NF-H
could affect microtubule stability through competition
with tau. Thus, a depletion of NF-H levels might yield more binding sites for tau on the surface of microtubules
resulting into extra stabilization of axonal microtubules.
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Increased Velocity of Neurofilament Transport in Absence of NF-H
Since overexpression of mouse or human NF-H genes in
transgenic mice slowed axonal transport of neurofilaments
(Collard et al., 1995; Marszalek et al., 1996
), we examined
whether the velocity of axonal transport of NF-L and NF-M
proteins would be increased in NF-H-deficient mice. This
was done by the injection of [35S]methionine into the L4-L5 spinal cord with subsequent analysis after 30 d of incorporated radioactivity for NF-L and NF-M proteins in 3-mm
segments of the sciatic nerve. In normal mice, at 30 d after
injection, the peak of incorporated radioactivity for the NF-L and NF-M proteins occurred in the 9-mm segment
of the sciatic nerve while the leading edge of radiolabeled
neurofilament proteins was found at the 15-mm segment
corresponding to a velocity of ~0.5 mm/d (Fig. 7). In contrast, for the NF-H
/
mice, the leading edge of radiolabeled NF-L and NF-M proteins was found at the 21-mm segment implying a velocity of ~0.7 mm/d (Fig. 7 C). To
facilitate comparison, the signals were quantified by image
analysis and converted to percentage of total radioactivity
(Fig. 7, D-F). Clearly, the peaks of radiolabeled NF-L and
NF-M proteins are broadened in the NF-H
/
axons as
compared with NF-H +/+ axons. Whereas <5% of radiolabeled NF-L and NF-M proteins were detected in the
15-mm sciatic segment of normal mice, ~25% of newly
synthesized NF-L and NF-M migrated to the 15-mm segment in NF-H
/
mice. In contrast, no dramatic changes
in the velocity of NF-M and NF-L transport occurred in
the NF-H +/
mice (Fig. 7, B and E).
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Relief of Motor Axonopathy Induced by IDPN
IDPN is a toxic agent known to segregate microtubules
from neurofilaments resulting in the excessive accumulation of neurofilaments in the proximal part of the axon
and with microtubules forming a central grouping in axons
(Griffin et al., 1978; Parhad et al., 1988
). The availability of
NF-H knockout mice provided us a unique opportunity to
assess the role of NF-H in axonopathy induced by this
neurotoxin. Intoxication of normal mice by IDPN led to
the formation of prominent neurofilamentous swellings in
proximal axons originating from spinal motor neurons
(Fig. 8, B and C). In contrast, after IDPN treatment, littermates heterozygous (Fig. 8, E and F) and homozygous
(Fig. 8, H and I) for the NF-H null mutation did not develop such axonal swellings in motor axons.
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Electron microscopic studies of transverse sections of
the L5 ventral root axons within the DRG, 7 d after IDPN
intoxication, revealed the absence of IDPN-induced segregation of microtubules from neurofilaments in NF-H /
mice (Fig. 9). We conclude from these results that NF-H is
a key mediator of motor axonopathy induced by IDPN exposure.
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Discussion |
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Our analysis of NF-H knockout mice provides direct evidence for roles of NF-H protein as a determinant of microtubule density in axons, as a modulator of velocity of
neurofilament protein transport in axons and as a key mediator of axonopathy induced by IDPN intoxication. A
number of previous reports suggested that neurofilaments
can interact between each other and with microtubules via
the COOH-terminal domains of NF-H or NF-M but the
significance of such interactions has remained elusive
(Hisanaga et al., 1990, 1993
). The presence of highly
charged amino acids in the long tail domains of NF-H also
led to the suggestion that this subunit would be implicated
as regulator of neurofilament packing density and perhaps
control axonal caliber (deWaegh et al., 1992
; Hsieh et al.,
1994
; Nixon et al., 1994
). Evidence for this notion has come from studies on the dysmyelinating mutant Trembler
mouse and on hypomyelinating transgenic mice expressing
in Schwann cells either a diphtheria toxin A or SV-40 large
T antigen in which reduced levels of NF-H phosphorylation resulted in decreases in axon caliber (deWaegh et al.,
1992
; Cole et al., 1994
). Therefore, as the NF-H subunit is
dispensable for the formation of a neurofilament network,
it is rather surprising that the disruption of the NF-H gene
had so little effects on the spacing density of neurofilaments and caliber of axons. These results can reflect in
part the existence of compensatory mechanisms. For instance, it is plausible that an hyperphosphorylation of the
NF-M subunit, as revealed by stronger immunodetection
signals with the SMI-31 antibody (Fig. 1 D), can compensate in part for NF-H function. Unlike NF-H
/
mice,
the NF-M
/
mice have decreased levels of NF-L and
dramatic reduction of axonal calibers (Elder et al., 1998
;
our unpublished observations). Therefore, it is clear that
the NF-H and NF-M proteins are not playing equivalent
roles in neurofilament function. The combined studies
with NF-M
/
and NF-H
/
mice indicate that NF-M
rather than NF-H protein is a key modulator of axonal caliber.
Another unexpected consequence of NF-H gene disruption was the dramatic increase of ~2.4-fold in the density
of microtubules in ventral root axons (Fig. 3 D). This was
further corroborated by similar increases in levels of assembled acetylated tubulin detected in cytoskeletal preparations from the sciatic nerve (Fig. 5). How could microtubule density in axons be regulated by the NF-H subunit?
One possibility, is that the absence of NF-H could somehow increase the rate of axonal transport of tubulin protein or of preassembled microtubules from the cell body.
However, the rise in assembled microtubules in NF-H /
axons was not accompanied by a corresponding increase in
the levels of total axonal tubulin (Fig. 1 D). This suggests
that the lack of NF-H contributed to increase the stability
of microtubules. One plausible mechanism by which NF-H
could affect microtubule stability is through competition with tau, a microtubule-associated protein (MAP) that
suppresses microtubule dynamics (Miyasaka et al., 1993
).
In vitro binding affinity assays demonstrated that both hypophosphorylated NF-H and tau, compete for similar
binding sites on the COOH-terminal region of tubulin
(Miyasaka et al., 1993
). Therefore, it is conceivable that a
depletion of NF-H levels might yield more binding sites for tau on the surface of microtubules resulting into extra
stabilization of axonal microtubules. This view is supported by the increased immunodetection of low molecular weight tau with TAU-1 mAb on immunoblots of
cytoskeletal preparations from the sciatic nerve of NF-H
/
mice (Fig. 6).
Recent studies with transgenic mice overexpressing murine NF-H led to the suggestion that NF-H can influence
the rate of neurofilament transport in axons (Marszalek et
al., 1996). The axonal transport studies shown here are
consistent with this view. The absence of NF-H increased
the velocity of axonal transport of NF-M and NF-L proteins by ~0.2 mm/d (Fig. 7). While this is not a trivial change, this is much less than an eightfold change of velocity suggested by studies of slow axonal transport during
development (Willard and Simon, 1983
). The exact molecular mechanism by which NF-H can affect neurofilament
transport remains unknown. It has been proposed that the
NF-H could shift an equilibrium toward assembly of stationary neurofilaments. The effect of NF-H on the mobility of neurofilament proteins could be indirect. The NF-M and NF-H proteins can compete for heterodimer formation with NF-L (Athlan and Mushynski, 1997
). Overexpressing NF-H provoked decreases in NF-M levels (Collard et al., 1995
; Marszalek et al., 1996
) while the absence
of NF-H levels resulted in a ~20% increase in NF-M levels. Therefore, one possibility is that changes in levels NF-H
affect the levels of NF-M which in turn could be a key regulator of NF-L transport. Future axonal transport studies
with NF-M knockout mice and double NF-H; NF-M
knockout mice should help to verify this hypothesis.
The IDPN intoxication studies described here further
support the view that neurofilaments and microtubules are
highly interdependent structures and that the NF-H subunit plays a key role in mediating these interactions. It is
remarkable that motor axonopathy due to IDPN intoxication was mitigated not only in NF-H /
mice but also in
NF-H +/
mice. Thus, a ~50% decrease in the levels of NF-H protein was sufficient to alter the properties of axonal cytoskeleton and to confer resistance to IDPN intoxication even though little effect was observed in axonal
transport. A key role for NF-H as a mediator of IDPN toxicity is consistent with the report of a transient changes in
the phosphorylation state of this protein preceding the onset of segregation of neurofilaments from microtubules
and neurofilament transport impairment (Tashiro et al.,
1994
). While NF-H is clearly acting as a key mediator of
IDPN-induced axonopathy, other molecules involved in
the phenomenon remain to be defined. Some of these factors could be cell-type specific as neurofilamentous swellings were still detectable in DRG sensory axons of both
NF-H +/
and NF-H
/
after IDPN treatment while
motor axons were exempt of swellings (data not shown).
Our finding that NF-H is a modulator of microtubule
density and a key mediator in IDPN-induced axonopathy
may be of relevance to neurodegenerative diseases involving cytoskeletal abnormalities. The abnormal neurofilament depositions in perikaya and neuronal processes are
frequently observed in neurodegenerative diseases such as
ALS (Carpenter, 1968; Chou, 1992
; Hirano et al., 1984
), Parkinson's disease (Schmidt et al., 1991
) and toxic neuronopathies induced by neurotoxin exposure (Asbury and
Johnson, 1978
; Graham et al., 1984
; Griffin et al., 1978
;
Troncoso, 1992). Transgenic mouse studies provided compelling evidence that a disorganization of the neurofilament network can play a causative role in the selective degeneration of motor neurons (Côté et al., 1993
; Xu et al.,
1993
; Lee et al., 1994
). Studies with transgenic mice overexpressing human NF-H led to the suggestion that the toxicity of disorganized neurofilaments is due to a general disruption of axonal transport (Collard et al., 1995
). While
transport impairment has been interpreted as reflecting a
physical block by neurofilament accumulations, the data
presented here suggest that changes in NF-H levels can
also perturb the dynamics and function of microtubules, which are key organelles of intracellular transport.
It is noteworthy that the lack of NF-H also resulted in a
decreased levels of assembled actin in extracts form the
sciatic nerve (Fig. 5 A). The NF-H protein was recently
found to be connected to actin filaments in sensory neurons by a linker protein encoded by a neuronal splice form
of the BPAG1 gene (Yang et al., 1996), which is responsible for the autosomal recessive dystonia musculorum. The
large primary sensory axons in BPAG1 knockout mice degenerate within 4 wk after birth, clearly demonstrating
that perturbation of the neurofilament-actin connections
may have profound effects on axonal integrity. Future
breeding studies with the NF-H
/
mice described here
will provide a unique approach to assess the in vivo contribution of NF-H protein to BPAG1-mediated degeneration and to other neurodegenerative diseases such as motor neuron disease.
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Footnotes |
---|
Received for publication 5 February 1998 and in revised form 25 June 1998.
The technical assistance of P. Hince, D. Altshuller, D. Houle, and G. Gagnon is gratefully acknowledged.
Address all correspondence to Jean-Pierre Julien, The Montreal General
Hospital Research Institute, 1650 Cedar Avenue, Montréal, Québec, Canada H3G 1A4. Tel.: (514) 937-6011 ext. 2361. Fax: (514) 934-8265. E-mail:
mdju{at}musica.mcgill.ca
This work was supported by the American Health Assistance Foundation and the Amyotrophic Lateral Sclerosis (ALS) Association (USA). Q. Zhu was a recipient of a fellowship from the NeuroScience Network, and J.-P. Julien has a MRC senior scholarship.
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Abbreviations used in this paper |
---|
ALS, amyotrophic lateral sclerosis;
DRG, dorsal root ganglia;
ES, embryonic stem;
IDPN, ,
'-iminodipropionitrile;
NF-H/NF-L/NF-M, neurofilament heavy/neurofilament light/
neurofilament medium.
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