Department of Pathology, Columbia University College of Physicians & Surgeons, 630 West 168th Street, New York, NY 10032, USA
* Author for correspondence (e-mail: rkl2{at}columbia.edu)
Accepted 4 September 2002
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Summary |
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Key words: Charcot-Marie-Tooth, Intermediate filament, Neurofilament, Assembly
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Introduction |
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Genetic linkage analyses have mapped a number of chromosomal loci linked to
CMT2 and so far three genes have been identified. Two independent studies have
demonstrated an association between mutations in the neurofilament light (NFL)
gene and CMT2, thus defining a new disease subtype called CMT2E. In both
families, the mutations have autosomal dominant patterns of inheritance
(Mersiyanova et al., 2000;
De Jonghe et al., 2001
). Two
additional genes have been linked to CMT2. A mutation with an autosomal
dominant pattern of inheritance in the gene encoding the motor protein
KIF1Bß has been linked to CMT2A (Zhao
et al., 2001
) and a mutation in the gene for lamin A/C, an
intermediate filament (IF) protein that is a constituent of the nuclear
envelope has been linked to an autosomal recessive form of CMT2
(De Sandre-Giovannoli et al.,
2002
).
Neurofilaments (NFs) are neuronal-specific IFs with important roles in the
development and maintenance of axonal structure
(Liem, 1993). NFs are
particularly abundant in large myelinated axons and are formed by three
subunits, described as high (NFH), medium (NFM), and light (NFL), according to
their molecular weights. Like other IF proteins, NF proteins have a tripartite
structure, with a central
-helical coil-coiled rod domain that mediates
dimerization, and globular N-terminal head and C-terminal tail domains. Rat
and mouse NFs are obligate heteropolymers in vivo, requiring the NFL subunit
and either the NFM or NFH subunit for the formation of extensive filamentous
networks in the absence of other cytoplasmic IFs
(Ching and Liem, 1993
;
Lee et al., 1993
). In
contrast, human NFL has been reported to self-assemble into a filamentous
network (Carter et al., 1998
).
Mice null for NFL are viable but display a 15-20% reduction in the number of
myelinated axons at 2 months of age. They also showed a reduction of axonal
diameter and delayed nerve regeneration
(Zhu et al., 1997
).
In order to investigate the effects of the CMT mutations on the assembly of
NFs, we have conducted transient transfection experiments with wild-type and
mutant rat and human NFL cDNAs. The reported mutations are a proline to
arginine change in the head domain (P8R) and a glutamine to proline change in
the rod domain (Q333P) of the human NFL subunit. A variant (D469N) in the tail
domain of NFL has been described while searching for mutations in NFs in
amyotrophic lateral sclerosis patients. This variant was not linked to disease
(Vechio et al., 1996) and we
used it as a control.
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Materials and Methods |
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The expression constructs for the hNFL variant (D469N), hNFL P8R mutant and hNFL Q333P mutant cDNAs (Fig. 1B) were generated by site-directed mutagenesis using the QuickChange Mutagenesis Kit (Stratagene). The sequences of the resulting clones were confirmed by sequencing. To facilitate simultaneous expression of wild-type and mutant NFL, we generated pCI-hNFL(Q333P)-hNFL(wt) by cloning the PCR fragment that contained the CMV promoter, hNFL(wt) cDNA and SV40 polyA signal into the BamHI site of pCI-hNFL(Q333P). The full-length cDNA of the wild-type hNFM gene was cloned by PCR from a human brain cDNA library (Clontech) using primers hNFM-5' (5'-gatgagctacacgttggactcgctgggcaa-3') and hNFM-3' (5'-ttagtcactctgggtgacttcctttactat-3') into the pCR2.1-TOPO vector (Invitrogen), and subsequently cloned into the EcoRI site of the pCI vector (Promega). Our clone contained the previously reported Val482Ala polymorphism in the sequence of human NFM (GenBank accession no. NM005382).
Transient transfections
We used two human adrenal carcinoma cells lines described by Sarria et al.
(Sarria et al., 1990). SW13
Vim+ cells express endogenous vimentin, while SW13 Vim-
cells are devoid of all cytoplasmic IFs. We maintained the cell lines in
DMEM-F12 media supplemented with 10% or 5% fetal bovine serum, respectively,
and 1% antibiotic solution (either penicillin-streptomycin or
antibiotic-antimycotic) at 37°C and 5% CO2. Transient
transfections were carried out in serum free medium using the GenePORTER
system (Gene Therapy Systems) as described in the manufacturer's protocol. For
indirect immunofluorescence microscopy, cells were grown on 18-mm glass
coverslips. 48 hours after transfection, cells were fixed in methanol at
-20°C for 10-15 minutes. and subjected to immunostaining. For western-blot
analysis, cells were harvested also at 48 hours post-transfection.
Indirect immunofluorescence microscopy
Immunofluorescence staining started with a blocking step using 10% normal
goat serum followed by incubation with the primary antibodies. After washing
with PBS, the cells were incubated with appropriate Alexa-Fluor-488 and -594
conjugated secondary antibodies (Molecular Probes) and washed again with PBS.
Finally, the coverslips were mounted on slides using Aquamount (Lerner
Laboratories) and visualized using a Nikon Eclipse 800 immunofluorescence
microscope and a Spot digital camera.
Western blotting
Preparation of cytoskeletal protein extracts using a protease inhibitor
cocktail (Roche) was carried out as previously described
(Ching and Liem, 1993). The
protein fractions were loaded on SDS-PAGE gels and transferred onto PVDF
membranes. After a blocking step in PBS-0.05% Tween20 (PBS-T) plus 5% nonfat
dry milk, the blots were incubated with primary antibodies in PBS-T plus 5%
nonfat dry milk, washed with PBS-T and incubated with 1:10,000 dilution of
HRP-conjugated goat anti-rabbit secondary antibody. After further washing with
PBS-T, protein detection was carried out using the ECL Detection System
(Amersham).
Antibodies
The following primary antibodies were used: mouse anti-NFL monoclonal
antibody (clone NR4, Sigma), mouse anti-NFH monoclonal antibody (clone N52,
Sigma), mouse anti-NFM monoclonal antibody (clone NN18, Sigma), rabbit
anti-NFL and anti-NFM polyclonal antibodies
(Kaplan et al., 1991), mouse
anti-vimentin monoclonal antibody (clone V9, Sigma), and mouse
anti-ß-tubulin monoclonal antibody (clone 2-28-33, Sigma).
Statistical analysis
The statistical analysis of the phenotypes observed with transient
transfection of the different hNFL cDNAs was carried out using the GraphPad
Prism version 3 software. We scored the phenotypes observed in the cells from
at least two transient transfection experiments in each case. We compared the
percentage of cells displaying each phenotype
(Table 1) using an unpaired
t-test when comparing two groups, and with one-way analysis of
variance (ANOVA) followed by the Bonferroni's Multiple Comparison Test when
more than two groups were compared. The level of significance was set in all
cases at P<0.05.
|
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Results |
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Effects of rNFL mutations in NF assembly
The two amino acids that are mutated in CMT2 patients and the one found as
a polymorphic variant are conserved between human and rat
(Fig. 1A). When either
wild-type or the D469N variant rNFL cDNAs were co-transfected in SW13
Vim- cells with rNFM or rNFH
(Fig. 2), the formation of a
normal filamentous network was observed. In all subsequent experiments, we
never observed a difference between the D469N variant and wild-type NFL. When
the P8R mutant rNFL cDNA was co-transfected with rNFM or rNFH, we observed two
different phenotypes. Most transfected cells had a filamentous network that
tended to bundle (Fig. 3A,B),
while in a few cells, the mutant rNFL co-localized with rNFM in cytoplasmic
aggregates and no filamentous network was observed
(Fig. 3C,D). In contrast, the
Q333P mutant rNFL formed aggregates that co-localized with the transfected
rNFM (Fig. 3E,F). These
aggregates varied in size and distributed throughout the cytoplasm. When the
mutant rNFL cDNAs were co-transfected with rNFH, similar results were
obtained. The P8R rNFL mutant co-assembled with rNFH into bundles
(Fig. 3G,H) or small
cytoplasmic aggregates (Fig.
3I,J), whereas the Q333P rNFL mutant formed small aggregates with
rNFH resulting in punctate staining (Fig.
3K,L). These results indicate that the CMT2 mutations are able to
influence the ability of rNFL to form an intact filamentous network in
transfected cells, with the rod mutation having a more dramatic effect
(disrupting filament formation) than the head mutation (formation of abnormal
thick and bundled filaments).
|
|
Effects of CMT2 mutations on hNFL self-assembly
Human NFL has been reported to form homopolymers in vivo, as opposed to rat
or mouse NFL (Carter et al.,
1998). The authors noted that there were eight differences between
rNFL and hNFL in the rod domain. Two of the differences between the rat and
human NFL sequences were reported to be important for the ability of hNFL to
form a homopolymeric filamentous network in SW13 Vim- cells; viz.
Arg161 (Gln in rat) and the absence of a serine (Ser251 in rat). However, when
we cloned the full-length cDNA for hNFL, we found that our cDNA clone had a
Gln in position 161, although rat Ser251 was also absent in this hNFL cDNA.
The sequence of our NFL cDNA was identical to the NFL draft sequence of the
Human Genome Project and also to a number of human EST clones (for more
details see Materials and Methods) indicating that the published sequence is
likely a variant of hNFL. The phenotypes observed upon single transfection of
our hNFL clones are summarized in Table
1. We found that both wild-type and D469N variant hNFL can form
homopolymers in SW13 Vim- cells
(Fig. 4A,B and data not shown).
We did not observe any statistically significant differences between the
phenotypes observed with wild-type hNFL and D469N hNFL
(Table 1A). These results
suggest that wild-type and D469N hNFL are phenotypically equivalent. An
extensive filamentous network was observed only in less than 10% of the
transfected cells (Fig. 4B).
The most prevalent phenotype was the presence of multiple short and thin
filaments (Fig. 4A), which in
half of the cells also contained a thicker filamentous core. In all these
cases, the cells could not be stained by anti-vimentin antibody, indicating
that the observed phenotypes were not due to the occasional revertants of
Vim- cells to Vim+ cells. Therefore, we confirmed that
unlike rodent NFL, hNFL is able to self-assemble into filaments, although the
hNFL networks were much less extensive than those formed by rNFL/rNFM or
rNFL/rNFH (Fig. 2). When we
transfected either the P8R (Fig.
4C) or the Q333P (Fig.
4D) hNFL mutants into SW13 Vim- cells, we found that
both mutations abolished the ability of hNFL to self-assemble into a
filamentous network. Instead, both mutant hNFL proteins formed aggregates in
transfected cells. These observations were statistically significant
(Table 1A). Small, dot-like
aggregates all around the cytoplasm were observed more frequently with the
Q333P hNFL mutant, while one huge aggregate occupying the entire cytoplasm of
the cell was observed more frequently with the P8R hNFL mutant.
|
Since both CMT2 NFL mutations show an autosomal dominant mode of
inheritance, we studied the effects of the CMT2-linked hNFL mutations on
wild-type hNFL assembly in transfected SW13 Vim- cells by
co-transfecting the wild-type hNFL with either the D469N variant, the P8R
mutant, or the Q333P mutant. Since we were not able to distinguish the
wild-type from the mutant hNFL with the available antibodies, we utilized
bicistronic hNFL constructs to assure that both wild-type and rod mutant NFL
were expressed in the same cells. We have used a similar approach for
co-expressing rNFL and rNFH (Leung et al.,
1999). The results of the experiment with the bicistronic
construct were identical to the results obtained with the co-transfection
experiments. Both the P8R mutant hNFL (Fig.
4E) and the Q333P mutant hNFL
(Fig. 4F) completely disrupted
the formation of wild-type hNFL filaments and caused the formation of
aggregates (Table 1B). In
contrast, co-transfection of wild-type hNFL with variant D469N hNFL resulted
in phenotypes similar to single transfections of either wild-type or D469N
hNFL (Table 1B).
Effects of CMT2 linked NFL mutations on co-assembly with hNFM
Although the gene for hNFM is tightly linked with the hNFL gene on
chromosome 8p21, no hNFM mutations have so far been described in CMT2
patients. Due to its tight linkage and developmentally coordinated expression
with hNFL, we selected hNFM to study the effects of hNFL mutations on the
formation of filaments in vivo. As observed for rodent NFM, hNFM was not able
to self-assemble into filaments (data not shown). The phenotypes observed upon
co-transfection of wild-type hNFM with the different hNFL clones are
summarized in Table 1C. Wild
type hNFL co-transfected with hNFM in SW13 Vim- cells resulted in
the formation of an extensive heteropolymeric filamentous network
(Fig. 5A,B). Similar results
were obtained with the D469N hNFL variant (data not shown). Transfection of
the Q333P mutant hNFL resulted in the formation of aggregates in transfected
cells (Fig. 6E,F). These
aggregates were distributed throughout the cytoplasm, but could also form
larger aggregates. In contrast, when hNFM was co-transfected with the P8R
mutant hNFL, we observed that the transfected cells displayed filaments that
were bundled, with some individual filaments splaying from the bundle
(Fig. 5C,D). Truncated thick
filaments could also be observed (cell on the left in
Fig. 5C,D). In addition, the
P8R mutant hNFL formed aggregates with hNFM in <5% of the transfected
cells. Nevertheless, we did not observe the formation of an extensive
filamentous network throughout the cytoplasm as in the case of co-transfected
wild-type or variant hNFL with hNFM (Fig.
5A,B). These studies confirm that wild-type and variant hNFL
co-transfected with hNFM form a normal filamentous network, whereas the
CMT2-linked NFL mutants have varying effects. The Q333P mutant showed a severe
ability to disrupt filament formation, resulting in aggregates, whereas the
P8R mutant showed a more subtle effect, with NFL/NFM co-polymers appearing as
bundled filaments, rather than as a filamentous network.
|
|
Effects of hNFL mutations on co-assembly with vimentin
All four hNFL clones were transfected in SW13 Vim+ cells in
order to assess the effects of hNFL mutations on the endogenous vimentin
network. Both wild-type hNFL (Fig.
6A,B) and D469N variant hNFL (not shown) co-assembled with the
endogenous vimentin network without causing any alterations. The P8R mutant
hNFL also co-localized with the endogenous vimentin, although the resulting
network was usually not as extensive as observed with the wild-type hNFL
(Fig. 6C,D). The effect of the
Q333P mutant hNFL on the endogenous vimentin network appeared to be dependent
on the relative amounts of vimentin and Q333P hNFL
(Fig. 6E-H). When low levels of
the Q333P mutant hNFL were expressed, it incorporated into the vimentin
network (Fig. 6E,F). As
expected due to the inability of the hNFL Q333P mutant to self-assemble, when
high levels of Q333P mutant hNFL are expressed, it does not appear to be able
to incorporate into the endogenous vimentin network
(Fig. 6G,H). Some cells
expressing very high levels of mutant protein displayed the co-localization of
vimentin with Q333P mutant hNFL in aggregates (not shown).
Effects of transfected hNFL on the microtubule network
NFs have been shown to associate with the microtubule network. In order to
investigate the possibility that the microtubules could be disrupted by the
overexpression of the mutant hNFL proteins, we transfected wild-type
(Fig. 7A), D469N variant
(Fig. 7B), P8R mutant
(Fig. 7C) and Q333P mutant
(Fig. 7D) hNFL constructs into
SW13 Vim- cells and co-stained the cells with anti-ß-tubulin
and anti-NFL antibodies. Overexpression of hNFL proteins (mutant or wild-type)
did not affect the endogenous microtubule network
(Fig. 7). Furthermore,
transfected SW13 Vim- cells treated with nocodazole to disrupt the
microtubule network also did not show any differences in the NFL networks
(mutant or wild-type).
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Discussion |
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The second NFL mutation (P8R) affects the non--helical head domain
of hNFL (De Jonghe et al.,
2001
). We used the Network Protein Sequence Analysis software
(Combet et al., 2000
) to
analyze the consensus secondary structure prediction for both mutant hNFL
proteins, but we did not find any differences due to both the P8R and the
Q333P mutations. Using the NetPhos 2.0 Prediction server
(Blom et al., 1999
) we
determined that the P8R mutation results in the appearance of a new potential
phosphorylation site (Ser11), for both protein kinase A and casein kinase-II.
Since phosphorylation of the head domain of NFL regulates its assembly and
disassembly (Nakamura et al.,
2000
; Sihag and Nixon,
1989
; Sihag and Nixon,
1991
), the possibility exists that changes in the phosphorylation
of the head domain of hNFL might be involved in the effects caused by the hNFL
P8R mutation in the assembly of IFs. Deletion analysis has demonstrated that
the head domain of NFL is required for filament assembly
(Ching and Liem, 1998
).
However, the contribution of individual amino acids of the head domain to
filament formation has not been fully determined. The P8R mutation had a less
severe effect on filament formation than the Q333P mutant NFL. Unlike
wild-type or variant hNFL, the P8R mutant hNFL was not able to self-assemble.
However, it co-assembled with hNFM into filaments, although these filaments
had a tendency to form bundles. Similar results were also obtained with the
corresponding mutation in rNFL. Moreover, the P8R mutant rNFL colocalized with
rNFM or rNFH in cytoplasmic aggregates in a small percentage of transfected
cells. Some studies have suggested that bundled NFs may undergo transport at a
slower rate than individual filaments
(Yabe et al., 2001
). Although
these studies were carried out using NFs that were highly phosphorylated in
the carboxyl-terminal domain, it is possible that this could also apply to the
case of the NF bundles caused by the P8R hNFL mutation.
Our transfection data suggest that both CMT2-linked mutations in NFL
influence the abilities of these proteins to assemble into filaments. Neither
human NFL mutant was able to self-assemble in SW13 Vim- cells. In
contrast, wild-type or variant hNFL generally self-assembled into short
filaments and more rarely into an extensive filamentous network. In
co-assembly studies aggregates were formed when either the rat or human Q333P
mutant NFL was co-expressed with the other NF proteins. The P8R mutant NFL
tended to form bundles of filaments or aggregates, rather than an extensive
filamentous network when co-transfected with NFM or NFH. We do not yet know
how these effects ultimately lead to neurodegeneration in CMT2E patients, but
it is possible that the resulting aggregated or bundled neurofilaments affect
the axoplasmic transport of the proteins. Recently, it was shown that
KIF1Bß+/- mice have a defect in transporting synaptic vesicle
precursors and suffer from progressive muscle weakness similar to human CMT2.
A mutation in the motor domain of KIF1Bß was identified in families
affected with CMT2A and previously assigned by linkage analysis to the
interval containing the KIF1Bß gene
(Zhao et al., 2001). We
believe the results described here will have an impact on the investigations
regarding the pathogenic mechanisms involved in CMT, and possibly in other
neurodegenerative diseases as well.
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Acknowledgments |
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