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The Journal of Neuroscience, July 15, 2000, 20(14):5321-5328
Formation of Intermediate Filament Protein Aggregates with
Disparate Effects in Two Transgenic Mouse Models Lacking the
Neurofilament Light Subunit
Jean-Martin
Beaulieu,
Hélène
Jacomy, and
Jean-Pierre
Julien
Centre for Research in Neurosciences, McGill University, The
Montreal General Hospital Research Institute, Montreal, Quebec, Canada
H3G 1A4
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ABSTRACT |
Protein aggregates containing intermediate filaments (IFs) are a
hallmark of degenerating spinal motor neurons in amyotrophic lateral
sclerosis (ALS). Recently, we reported that a deficiency in
neurofilament light subunit (NF-L), a phenomenon associated with ALS,
promoted the formation of IF inclusions with ensuing motor neuron death
in transgenic mice overproducing peripherin, a type III IF protein
detected in axonal inclusions of ALS patients. To further assess the
role of NF-L in the formation of abnormal IF inclusions, we generated
transgenic mice overexpressing human neurofilament heavy subunits
(hNF-H) in a context of targeted disruption of the NF-L gene (hH;L /
mice). The hH;L/ mice exhibited motor dysfunction, and they
developed nonfilamentous protein aggregates containing NF-H and
peripherin proteins in the perikarya of spinal motor neurons. However,
the perikaryal protein aggregates in the hH;L/ mice did not provoke
motor neuron death, unlike toxic IF inclusions induced by peripherin
overexpression in NF-L null mice (Per;L/ mice). Our results
indicate that different types of IF protein aggregates with distinct
properties may occur in a context of NF-L deficiency and that an axonal
localization of such aggregates may be an important factor of toxicity.
Key words:
neurofilament; peripherin; intermediate filament; transgenic mouse; ALS; amyotrophic lateral sclerosis; motor neuron
disease; neurodegeneration
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INTRODUCTION |
Amyotrophic lateral sclerosis (ALS)
is a late onset motor neuron disease characterized by the degeneration
of spinal motor neurons, leading to paralysis and death. Although
~3% of ALS patients have inherited mutations in the Cu/Zn superoxide
dismutase (SOD1) gene (Rosen et al., 1993 ), the causes of disease for
the vast majority of ALS patients remain unknown. Inclusion bodies,
also called spheroids, containing neuronal intermediate filaments (IFs) are a common feature of degenerating motor neurons in ALS (Carpenter, 1968; Corbo and Hays, 1992; Migheli et al., 1993). Such IF inclusions also occur in transgenic mice expressing ALS-linked SOD1 mutant proteins, suggesting a role for these inclusions in the pathology of
ALS (Tu et al., 1996; Beaulieu et al., 1999a). Adult motor neurons can
express up to five different IF proteins detectable in IF inclusions of
ALS patients (Tu et al., 1996; Corbo and Hays, 1992; Migheli et al.,
1993). These proteins are the neurofilament (NF) light (NF-L), medium
(NF-M), and heavy subunits (NF-H),  -internexin and peripherin
(Hoffman and Lasek, 1975; Pachter and Liem, 1985; Parysek and Goldman,
1988). Multiple cell transfection and transgenic mouse studies have
demonstrated that NFs are formed by the heteropolymerization of NF-L
with NF-M and/or NF-H (Lee et al., 1993; Ching and Liem, 1993; Zhu et
al., 1997; Jacomy et al., 1999). On the other hand, -internexin and
peripherin can either self-polymerize into IFs or interact with NF
proteins according to modalities that are not yet well understood
(Parysek et al., 1991; Ching and Liem, 1993; Athlan and Mushynski,
1997; Beaulieu et al., 1999a,b).
A 60% decrease in mRNA levels for NF-L has been reported in motor
neurons of ALS patients (Bergeron et al., 1994). Although the NF-L
knock-out genotype (NF-L/) is not pathogenic for mice (Zhu et al.,
1997), our recent studies showed that NF-L levels can affect the
progression of motor neuron pathologies caused by an upregulation of
other IF proteins. The overexpression of wild-type peripherin led to
the formation of IF inclusions with ensuing death of motor neurons in
2-year-old mice (Beaulieu et al., 1999a). Remarkably, the motor neuron
disease in peripherin transgenic mice was precipitated by a deficiency
of NF-L (Beaulieu et al., 1999a). Transgenic mice expressing a human
NF-H protein (hNF-H) also develop abnormal IF accumulations in spinal
motor neurons, with ensuing motor dysfunction during aging
(Côté et al., 1993). Again, in this mouse model, the
pathogenesis was altered by changes in NF-L levels. Thus, the
overexpression of a human NF-L transgene in hNF-H mice was able to
reduce the perikaryal NF swellings and rescue the motor neuronopathy
(Meier et al., 1999).
To further analyze the role of NF-L in the formation of neuronal IF
inclusions and in motor neuron disease, we generated transgenic mice
overexpressing hNF-H proteins in a context of NF-L deficiency. In
absence of NF-L subunits, the hNF-H transgenic mice developed nonfilamentous protein aggregates containing NF-H and peripherin proteins in the perikarya of spinal motor neurons. However, the protein
aggregates in the hH;L/ mice did not provoke motor neuron death,
unlike IF inclusion bodies resulting from the overexpression of
peripherin proteins in NF-L null mice. Our results suggest that axonal
localization is a factor that may contribute to the toxicity of IF
protein aggregates in motor neurons.
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MATERIALS AND METHODS |
Transgenic mice. The hH;L/ mice were obtained by
breeding the previously described hNF-H transgenic mice (line 200)
(Côté et al., 1993) with NF-L/ mice (Zhu et al., 1997).
The genotypes of the resulting mice were determined by Southern blot
analysis of mouse tail DNA according to previously described procedures (Côté et al., 1993; Zhu et al., 1997). All hNF-H transgenic mice used in this study were homozygous for the hNF-H transgene and in
a C57BL6 enriched background. The peripherin transgenic mice
overexpressing the wild-type mouse peripherin protein under the control
of the peripherin promoter (Per mice) and Per;L/ mice were
described previously (Beaulieu et al., 1999a). The use and maintenance
of mice described in this article were performed according to the
guidelines of Care and Use of Experimental Animals of
the Canadian Council on Animal Care.
Antibodies. The anti-human NF-H (OC95) and anti-mouse NF-H
(OC59) rat monoclonal antibodies were kind gifts from Dr. Virginia Lee
(University of Pennsylvania, Philadelphia, PA). The anti-NF-H hypophosphorylated (Smi-32) and hyperphosphorylated (Smi-31) monoclonal antibodies were from Sternberger Monoclonals (Lutherville, MD). The
anti-NF-H polyclonal antibody (N-4142) was from Sigma (Oakville, Canada). The anti-NF-M monoclonal antibody (NN-18), the anti-NF-L monoclonal antibody (NR-4), and the anti-actin clone c4 monoclonal antibody were from Roche Molecular Biochemical (Laval, Canada). The
anti-peripherin monoclonal antibody (MAB1527), the anti-peripherin polyclonal antibody (AB1530), and the anti -internexin polyclonal antibody (AB1515) were from Chemicon (Mississauga, Canada). The anti-MG160 polyclonal antibody (Croul et al., 1990) was a kind gift
from Dr. Nicolas K. Gonatas (University of Pennsylvania, Philadelphia, PA).
Western blot analysis. For total protein extraction, tissues
were homogenized in SDS-urea buffer (5 mg/ml SDS, 8 M urea). For isolation of the cytoskeletal
insoluble proteins, tissues were first homogenized at 4°C in a Triton
buffer containing 10 mM Tris, 150 mM NaCl, 1 mM EDTA, 1%
Triton X-100, and a cocktail of protease inhibitors (2 mM PMSF, 2 mg/ml leupeptin, 1 mg/ml pepstatin,
and 10 mg/ml aprotinin). Homogenates were centrifuged at 14,000 × g for 15 min at 4°C using a Sorvall MC-12V centrifuge (Dupont, Guelph, Canada). The supernatants (soluble fraction) were
collected and kept for further use. The pellets (insoluble fraction)
were resuspended in a volume of SDS-urea buffer equivalent to the
volume of the soluble fraction. For both types of extracts, the protein
concentration was measured using a DC-protein assay (Bio-Rad,
Mississauga, Canada). Proteins were separated on 10% SDS-PAGE and
transferred to nitrocellulose membranes. To ensure that equivalent
amounts of proteins were used for each sample, duplicate gels were
routinely stained with Coomassie blue-R250. Proteins were detected
using primary antibodies and peroxidase-conjugated anti-mouse IgG,
anti-rat IgG, or anti-rabbit IgG secondary antibodies from Jackson
ImmunoResearch (Mississauga, Canada). Immune complexes were revealed
using the Renaissance chemiluminescence reagents (NEN, Boston, MA).
Densitometric analysis were performed from autoradiograms using NIH
Image software version 1.62 for Power Macintosh (Apple Computers,
Cupertino, CA).
Partial proteolysis of NF-H. Partial proteolytic digestion
was performed following a modification of the method described by
Julien and Mushynski (1983). The spinal cord samples were homogenized in Triton buffer in the absence of protease inhibitor. Soluble and
insoluble protein fractions were prepared by centrifugation as
described above. The pellets were resuspended at a concentration of 3 mg/ml protein in 0.05 M
2(N-morpholino) ethanesulphonic acid, pH 6.5, and the
supernatants were diluted at the same protein concentration in the same
buffer. Both fractions were then digested separately with
-chymotrypsin (Roche Molecular Biochemical, Laval, Canada) at 30°C
for 0, 8, or 15 min with an enzyme-to-protein ratio of 1:3000. The
digestion was stopped by the addition of PMSF at a final concentration
of 2 mM. Each digestion product was then analyzed
by Western blotting.
Immunohistological analysis. Mice were anesthetized by
injection of chloral hydrate. For immunohistochemistry, mice were
perfused with a 16 gm/l sodium cacodylate buffer, pH 7.5, followed by
fixative (3% glutaraldehyde in sodium cacodylate buffer). Floating
50-µm-thick vibratome sections were rinsed in PBS, treated for 30 min
with a 10 mg/ml sodium borohydride solution, and then blocked for 1 hr
in a buffer containing 30 mg/ml BSA, 0.5% Triton X-100, and 0.3 mg/ml
hydrogen peroxide in PBS. Incubation with antibodies was performed
overnight at room temperature in a buffer containing 30 mg/ml BSA and
0.05% Triton X-100 in PBS. Labeling was done using a Vector ABC kit
(Vector Laboratories, Burlingame, CA) and Sigmafast tablets (Sigma).
For double indirect immunofluorescence, mice were perfused with PBS, pH
7.5, followed by 40 mg/ml paraformaldehyde in PBS. Tissues were
post-fixed for 2 hr in paraformaldehyde, rinsed in PBS, and incubated
overnight in a 200 mg/ml phosphate-buffered sucrose solution. Sections
of 10 µm were prepared using a cryostat. Sections were blocked for 30 min in PBS blocking buffer and incubated overnight with primary
antibodies. Immune complexes were revealed with anti-mouse FITC and
anti-rabbit rhodamine-conjugated antibodies (Jackson ImmunoResearch) at
a dilution of 1:200. Samples were mounted in Prolong (Molecular Probes,
Eugene, OR) and examined under a fluorescence microscope.
Composite overlaid pictures of double immunofluorescence were derived
from digitalized color films using Photoshop 4.0 for Macintosh
computers (Adobe Systems, San Jose, CA).
In situ detergent solubility assay. The spinal cords were
rapidly dissected out from unfixed mice, frozen at 80°C in Tissue Tek (Miles, Elkhart, IN), and cut in 10 µm sections using a cryostat. Sections were then incubated on slides at room temperature for 15 min
in Triton buffer (10 mM Tris, 150 mM NaCl, 1 mM EDTA, and 1%
Triton X-100) or in PBS, rinsed three times in PBS, and fixed for 30 min with a 4% paraformaldehyde phosphate-buffered solution. The
sections were then processed for double indirect immunofluorescence in
parallel with untreated sections obtained from mice fixed with a 4%
paraformaldehyde solution.
Light and electron microscopy. Tissues were prepared for
embedding in Epon as described by Zhu et al. (1997). Thin sections were
stained with toluidine blue and examined under a light microscope. Counting and morphological analysis of L5 ventral root axons was performed using an Image-1 analysis software (Universal Imaging, West
Chester, PA). For electron microscopy, sections were stained with a
lead citrate solution and examined with a Phillips CM10 transmission
electron microscope.
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RESULTS |
Absence of NF-L does not alleviate neuronopathy caused by
overexpression of hNF-H
The hH;L/ mice were derived by breeding procedures with hNF-H
transgenic mice (line 200) (Côté et al., 1993) and
NF-L/ mice (Zhu et al., 1997) to obtain mice that were homozygous
for both hNF-H transgene and NF-L disrupted gene
(hNF-H+/+;NF-L/). Western blots revealed reduced levels of mouse
and human NF-H proteins in the spinal cord of hH;L/ mice when
compared with homozygous hNF-H transgenic mice having a normal NF-L
genotype (Fig. 1A).
Similarly, a decrease of endogenous NF-H protein content also occurred
in NF-L/ mice. The reductions in levels of NF-H, NF-M, and to a
lesser extent in peripherin have been reported in NF-L/ mice and
are probably the result of an enhanced proteolytic turnover of
unassembled or disorganized IF proteins in the absence of NF-L (Zhu et
al., 1997; Williamson et al., 1998; Beaulieu et al., 1999a; Levavasseur
et al., 1999).
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Figure 1.
Motor dysfunction despite reduced levels of hNF-H
in hH;L/ mice. A, Western blot analysis of total
protein extracts (10 µg) from the spinal cord of 3-month-old control
(W), NF-L/ (L/),
hNF-H transgenic (hH), and hH;L/ mice.
Protein immunodetection was performed using the following antibodies:
hNF-H (OC95, 1:200 dilution), mouse NF-H (mNF-H)
(OC59, 1:200 dilution), NF-L (NR-4, 1:1000 dilution),
and actin (clone c4, 1:5000 dilution). B, Severe motor
dysfunction in a 2-year-old hH;L/ mouse.
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Despite overall reduction in levels of hNF-H protein, mice with the
hH;L/ genotype showed neuropathological dysfunctions similar to
those occurring in the hNF-H transgenic mice (Côté et al.,
1993). Both hNF-H transgenic mice and hH;L/ mice developed normally
and had no overt phenotypes within their first month of life. After
this period, they progressively developed tremors and muscle weakness
culminating in a severe impairment of hind limb functions at ~2 years
of age (Fig. 1B).
Nonfilamentous NF-H inclusions in the absence of NF-L
Light microscopy of thin sections from the lumbar spinal cord
stained with toluidine blue did not reveal inclusions in motor neurons
of adult normal mice (Fig.
2A). However, small
inclusions were observed in perikarya of some motor neurons from
NF-L/ mice (Fig. 2B). Large inclusions filling
nearly all of the perikarya were observed in most spinal motor neurons
of both hNF-H transgenic and hH;L/ mice (Fig.
2C,D). These inclusions were formed before 3 weeks of age and persisted throughout life. Except for their smaller
size, the inclusions of hH;L/ mice did not differ from those of
hNF-H transgenic mice when observed at light microscopy. Immunohistochemical staining with the OC95 antibody confirmed that the
inclusions in hNF-H transgenic mice and hH;L/ mice contained the
hNF-H protein (Fig. 2E). These inclusions were also detected with the Smi-31 and Smi-32 antibodies, indicating the presence
of both hyperphosphorylated (Smi-31) and hypophosphorylated (Smi-32)
NF-H epitopes (Fig. 2E; see Fig.
4A). Electron microscopy revealed that the inclusions
in hNF-H transgenic mice consisted of disorganized aggregates of 10 nm
filaments (Fig. 3A). In
contrast, the inclusions in hH;L/ mice were devoid of any filaments
(Fig. 3B,C). Despite this lack of
filaments, the inclusions in hH;L/ mice excluded other cytoplasmic
organelles, such as mitochondria and microtubules, suggesting that the
inclusions are not composed of freely diffusing proteins (Fig.
3C).
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Figure 2.
Perikaryal inclusions in the absence of NF-L
(A-D). Spinal cord sections stained with
toluidine blue show the presence of hyaline inclusions in the perikarya
of motor neurons (black arrowheads) from NF-L/
(B), hNF-H transgenic (C),
and hH;L/ (D) mice at 2 years of age. Such
inclusions were absent in motor neurons of normal (wt)
mice (A). E, Immunohistochemistry
revealing the presence of hNF-H (OC-95) and
phosphorylated NF-H epitopes (SMI-31) in inclusions of
both hH and hH;L/ mice. The scale bar in D applies to
A-D. Scale bars, 50 µm.
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Figure 3.
Absence of IF structures in perikaryal inclusions
of hH;L/ mice. A, B, Electron
micrographs show IF structures in inclusions of hNF-H transgenic mice
(A) and their absence in the inclusions of
hH;L/ mice (B). C, Electron
micrograph showing the segregation of microtubules (small
arrowheads) and mitochondria (large arrowheads)
in an inclusion of hH;L/ mouse. Scale bars: (in B)
A, B, 0.3 µm; C, 2 µm.
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The inclusions in hH;L/ mice are resistant to
Triton extraction
To further characterize the interactions of IF proteins in the
inclusions of hH;L/ mice, an in situ Triton
solubilization assay was performed on unfixed cryostat sections from
the spinal cord. The NF-H protein was detected using the monoclonal
antibody Smi-32, whereas a polyclonal antibody against the Golgi
apparatus-associated protein MG160 (Croul et al., 1990) was used as a
control for protein extraction. The Triton treatment successfully
abolished MG160 immunoreactivity, although it failed to remove NF-H
inclusions in sections prepared from the hNF-H transgenic and hH;L/
mice (Fig. 4A). This
suggested that some interactions still occur between IF proteins in the
inclusions of hH;L/ mice. Western blots of soluble and insoluble
spinal cord extracts from hNF-H transgenic and hH;L/ mice confirmed
that 30% of mouse NF-H and 16% of human NF-H protein existed in an
insoluble form in the hH;L/ mice (Fig. 4B). The
other neuronal IF proteins, NF-M, -internexin, and peripherin, were
mostly recovered in the insoluble fractions in hNF-H transgenic and
hH;L/ mice (Fig. 4B).
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Figure 4.
The inclusions in hH;L/ mice are
resistant to Triton extraction. Cryostat sections from unperfused
3-month-old hNF-H transgenic and hH;L/ mice were incubated for 15 min in either PBS or 1% Triton buffer. A double immunofluorescence
analysis demonstrates that the NF-H inclusions stained by Smi-32
immunoreactivity (arrowheads) are not extracted by this
treatment, whereas the Golgi apparatus membrane-associated protein
MG160 is extracted. Scale bar, 50 µm. B, Western blot of soluble
(S) and insoluble
(I) fractions (2.5 µg) of spinal cord
homogenates from 3-month-old hNF-H transgenic
(hH) and hH;L/ mice. Protein detection was
performed using the following antibodies: OC95 (1:200), hNF-H; OC59
(1:200), mouse NF-H and NF-M; NN-18 (1:1000), NF-L; NR-4 (1:1000),
peripherin (Per); MAB1527 (1:1000), actin
(Act); and clone c4 (1:5000) and -internexin
(-Int) AB1515 (1:2000).
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Assembly of the NF-H rod domain in hH;L/ mice
Because the first step of IF assembly involves the formation of
coiled-coil dimers between the rod domains of IF proteins (for review,
see Fuchs and Weber, 1994), we have performed partial -chymotrypsin
digestion to examine whether the rod domain of NF-H is involved in
protein-protein interactions in hH;L/ mice. Potential
-chymotrypsin cleavage sites (Fig.
5A) were identified from the
published sequence of the mouse (GenBank accession number P19246) and human (GenBank accession number P12036) NF-H proteins.
Multiple potential -chymotrypsin digestion sites exist in the head
and rod domain of both mouse and human NF-H proteins. It has been
demonstrated previously that -chymotrypsin treatment of unassembled
NF-H proteins leads to the formation of a single 160 kDa fragment
containing the tail domain of NF-H, whereas digestion of assembled NF-H
proteins generates additional fragments in the range of 40 kDa
corresponding to segments of the head and rod domain of NF-H, which are
protected from -chymotrypsin digestion because of assembly
into IF structures (Julien and Mushynski, 1983). Partial
-chymotrypsin digestions were performed on soluble and insoluble
fractions of spinal cord homogenates from either the hNF-H transgenic
or hH;L/ mice. Similar results were obtained with mice of these two
genotypes (Fig. 5B). Digestion of proteins from the soluble
fractions led to the formation of an NF-H fragment of ~160 kDa
detectable by Western blotting with either a polyclonal anti-NF-H
antibody or the anti-tail domain antibody Smi-32 (Sternberger and
Sternberger, 1983). Thus, this large 160 kDa fragment contained the
non-digested tail domain of NF-H proteins. The same large fragment was
also obtained after -chymotrypsin digestion of the insoluble
fraction of homogenates. However, staining of Western blots from the
insoluble protein digestion products with the anti-NF-H polyclonal
antibody revealed additional NF-H immunoreactive fragments in the range
of 40-50 kDa. These fragments were not detected by Smi-32 staining,
indicating that they are not derived from the tail domain of NF-H and
that they correspond to protected fragments from the head and rod
domains. These results further suggest that the NF-H rod domain is
involved in the formation of insoluble protein aggregates.
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Figure 5.
Partial -chymotrypsin digestion of insoluble
NF-H. A, Potential -chymotrypsin digestion sites
(black arrowheads) along the head and rod domains of
both mouse (mNF-H) and human
(hNF-H) NF-H proteins. Black boxes
indicate the -helical segments of NF-H rod domain. B,
Western blot analysis of the -chymotrypsin digestion products.
Soluble and insoluble fractions from spinal cord homogenates of hNF-H
transgenic or hH;L/ mice were treated with -chymotrypsin for 0, 8, and 15 min as described in Materials and Methods. The
immunodetection of NF-H digestion fragments was performed using an
anti-NF-H polyclonal antibody (N-4147, 1:2000 dilution) or the Smi-32
(1:500 dilution) monoclonal antibody, which is specific to
unphosphorylated (Lys/Ser/Pro) residues present in the tail domains of
both human and mouse NF-H proteins. Digestion of soluble NF-H proteins
from both mouse models led to the formation of a single large fragment
immunoreactive for both Smi-32 and NF-H polyclonal antibody, whereas
digestion of insoluble NF-H proteins led to the formation of additional
NF-H fragments (black arrowheads) that were only
immunoreactive with the polyclonal antibody. Numbers on
the left of gels in B correspond to
molecular weight markers in kilodaltons.
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NF-H colocalizes with peripherin in inclusion bodies of
hH;L/ mice
We have searched for potential interaction partners of NF-H in
inclusions bodies from the hH;L/ mice. In the absence of NF-L, the
NF-H protein can form heterodimers or interact in vivo with
-internexin and peripherin, although the interaction of peripherin
with NF-H does not lead to the formation of a normal IF network (Ching
and Liem, 1993; Athlan and Mushynski, 1997; Beaulieu et al., 1999a,b).
Double immunofluorescence staining was performed using the Smi-32
monoclonal antibody and polyclonal antibodies directed against
-internexin or peripherin. The results demonstrate the presence of
peripherin in most inclusions of hH;L/ mice, whereas -internexin
was only rarely detected in these same inclusions (Fig.
6).
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Figure 6.
Colocalization of NF-H with peripherin but
not with -internexin in hH;L/ mice. The NF-H protein was
detected (green) using the mouse monoclonal
antibody Smi-32 (1:500). Polyclonal antibodies
(red) were used to detect peripherin
(Per; AB1530, 1:5000) and -internexin
(-Inter; AB1515, 1:100). Composite images were
obtained by overlaying the green and red
images. Scale bar, 100 µm.
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Disparate effects of NF-L deficiency in hNF-H and peripherin
transgenic mice
Light microscopy of thin sections from the L5 ventral roots
revealed a reduction of axonal caliber for the hNF-H transgenic, NF-L/, and hH;L/ mice (Fig.
7A-E). However, the extent of axonal atrophy differs for each genotype. The hNF-H transgenic mice
were the least affected by the reduction of axonal caliber, followed by
the NF-L/ and hH;L/ mice, which exhibited the most severe
atrophy of axons (Fig. 7E). Despite robust motor dysfunction and axonal atrophy in hNF-H transgenic and hH;L/ mice, no massive degeneration of axons was observed in 2-year-old mice of either genotype (Fig. 7A-D). In agreement with our previous
observations with NF-L/ mice (Zhu et al., 1997; Beaulieu et al.,
1999a), there was a similar ~20% loss of L5 ventral roots axons in
2-year-old mice from the NF-L/ and hH;L/ genotypes (Fig.
7F). In contrast, 6-month-old Per;L/ mice
suffered a 46% loss of their motor neurons, whereas the peripherin
transgenic mice having normal NF-L levels did not suffer axonal loss at
this age (Fig. 7F). These results show that an NF-L
deficiency enhances the detrimental effects of peripherin
overexpression but not of hNF-H overexpression.
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Figure 7.
hNF-H overexpression exacerbates axonal atrophy
but not axonal loss in NF-L null mice. A-D, Toluidine
blue-stained sections of the L5 ventral roots from control
(A), hNF-H transgenic (B),
NF-L/ (C), and hH;L/
(D) mice. Note that little axonal degeneration
occurred in the hNF-H transgenic and hH;L/ mice. E,
Caliber distribution of L5 ventral root axons in normal
(WT), hNF-H transgenic
(hH), NF-L/ (L/), and
hH;L/ mice. F, Average number of axons in L5 ventral
roots of mice. These counts were performed on 2-year-old normal
(wt), hNF-H transgenic (hH),
NF-L/ (L/), and hH;L/ mice and on
6-month-old Per;L/ and peripherin transgenic mice
(Per). Scale bar, 5 µm. Error bars indicate SD.
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Axonal but not perikaryal IF inclusions correlate with motor
neuron death
Both the hH;L/ and Per;L/ mice develop IF inclusions
containing peripherin and NF-H (Fig. 6) (Beaulieu et al., 1999a). However, only the Per;L/ mice suffer motor neuron death.
Immunohistochemistry and light microscopy of spinal cord sections from
3-month-old hH;L/ and Per;L/ mice revealed major differences in
the size and distribution of peripherin-containing inclusions between
mice of these two genotypes (Fig.
8B-E). Motor neurons
of hH;L/ mice were characterized by the formation of a single large
accumulation that filled most of the perikaryon with occasional filling
of the proximal axons. In contrast, motor neurons of Per;L/ mice contained multiple small inclusions that were found in both perikarya and axons. Western blots of soluble and insoluble protein preparations from the spinal cord of 3-month-old NF-L/, hH;L/ and Per;L/ mice revealed that peripherin was totally insoluble in these three types of mice. However, peripherin was much more abundant in the Per;L/ mice (Fig. 8A).
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Figure 8.
Peripherin detection in protein aggregates of
hH;L/ and Per;L/ mice. A, Western blot of
soluble and insoluble fractions of spinal cord homogenates from
NF-L/ (L/), hH;L/, and Per;L/ mice. The
insoluble protein fraction (2.5 µ g) and an equal
volume of the soluble protein fraction were loaded on gel. Protein
detection was performed using the following antibodies: peripherin
(P); MAB1527 (1:1000), actin
(A); and clone c4 (1:5000). B,
C, Immunohistochemical detection of peripherin in the
spinal cord with a polyclonal antibody (AB1530, 1:5,000) showing the
difference in the size and distribution of peripherin-containing
inclusions (arrowheads) between hH;L/
(B) and Per;L/ (C)
mice. D, E, Thin sections stained with
toluidine blue showing a large hyaline inclusion filling the perikaryon
of a motor neuron in an hH;L/ mouse (D) and a
typical small hyaline inclusion (arrowhead) in the
perikaryon of a motor neuron in a Per;L/ mouse
(E). Scale bars: C, 100 µm;
E, 10 µm.
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DISCUSSION |
The presence of IF protein aggregates in motor neurons is a common
pathological finding in human ALS (Carpenter, 1968) (for review, see
Chou, 1995), but it appears paradoxical that the levels of NF-L mRNA
are reduced in this disease (Bergeron et al., 1994). The results
presented here demonstrate that the formation of IF protein aggregates
in motor neurons may occur in a context of NF-L deficiency. However, in
the absence of NF-L, different types of IF protein aggregates with
distinct properties were formed by the overexpression of different IF transgenes.
The overexpression of hNF-H proteins in NF-L null mice led to formation
of protein aggregates in the perikarya of motor neurons. These
perikaryal aggregates in hH;L/ mice were not as large as those
occurring in hNF-H transgenic mice having normal NF-L levels, and they
were not composed of 10 nm filaments. The lack of NF-L caused a
reduction in total NF-H content. The decreased NF-H levels are likely
the result of increased proteolytic degradation of unassembled NF-H
proteins in the absence of NF structures (Zhu et al., 1997). Yet, a
fraction of NF-H proteins escaped degradation and formed insoluble
protein aggregates in the hH;L/ mice. Although IF structures were
not detected in these aggregates, our results suggest that the NF-H
protein was present in the form of oligomeric structures. Thus, the
NF-H rod domain in the insoluble protein fraction was protected from
partial digestion by -chymotrypsin (Fig. 5), indicating the
formation of coiled-coil structures involving the rod domain of NF-H.
Moreover, the colocalization of peripherin with NF-H in the protein
aggregates of hH;L/ mice suggests the existence of oligomeric
structures made up of NF-H together with peripherin. This view is in
agreement with reports that peripherin can form heterodimers with NF-H
(Athlan et al., 1997) and that NF-H and NF-M can prevent the formation
of a normal peripherin IF network in transfected cultured cells and
transgenic mice (Beaulieu et al., 1999a,b).
The overexpression of peripherin in NF-L null mice also resulted in the
development of IF protein aggregates. However, the aggregates in
Per;L/ mice are composed of disorganized 10 nm filaments, unlike
aggregates in hH;L/ mice that are nonfilamentous. The different
types of IF protein inclusions and their distinct localization in these
two mouse models are basically the result of different ratios of
peripherin to NF-H proteins. At a low peripherin-to-NF-H ratio
occurring in the hH;L/ mice, the assembly of peripherin into IF
structures was impeded by excess NF-H, preventing the formation of a 10 nm filament network. Such destabilization of peripherin by NF-H has
been observed previously in SW13vim() cells cotransfected with
peripherin and NF-H expression vectors (Beaulieu et al., 1999b). On the
other hand, at a high peripherin-to-NF-H ratio as in the Per;L/
mice, peripherin proteins were able to form disorganized filaments with
NF-H and move into motor axons (Beaulieu et al., 1999a).
The mouse models described here provide good examples of distinct
phenotypes produced by different types of IF protein aggregates. The
small IF aggregates in the Per;L/ mice are very toxic and provoke
progressive motor neuron death. In contrast, the large perikaryal
swellings in motor neurons of hNF-H;L+/+ or hNF-H;L/ transgenic
mice are relatively well tolerated. These hNF-H transgenic mice develop
motor dysfunction, but they do not suffer motor neuron death.
Alterations in the electrophysiological properties of peripheral nerves
detected in the hNF-H transgenic mice are likely responsible for such
motor dysfunction (Kriz et al., 2000).
The toxic effects of IF aggregates in the Per;L/ mice, which have
morphological features similar to inclusion bodies in human ALS, may be
related in part to the sequestering of organelles such as mitochondria
(Beaulieu et al., 1999a). On the contrary, cellular organelles were
segregated from the perikaryal protein aggregates of the hNF-H;L/
mice (Fig. 3). It is also possible that the location of IF protein
aggregates in the axon constitutes an important determinant of
toxicity. As shown in Table 1, which summarizes the various transgenic mouse models with IF abnormalities, there is one example of partial motor neuron loss associated with massive, strictly perikaryal, IF inclusions. The NF-H/ -galactosidase transgene resulted in large perikaryal NF accumulations, a scarcity of
axonal IF structures comparable with the one observed in NF-L/ mice, and in a 20% motor neuron loss (Eyer and Peterson, 1994; Tu et
al., 1997). However, it is noteworthy that the massive death of spinal
motor neurons occurred only in mouse models with multiple IF inclusions
in motor axons, i.e., in mice expressing a mutant NF-L protein (Lee et
al., 1994) or peripherin transgenes (Beaulieu et al., 1999a). Such
axonal IF inclusions could block axonal transport, a mechanism of
neurodegeneration referred as the "strangulation model" (for
review, see Julien, 1997; Cleveland, 1999).
The results presented here demonstrate that an NF-L deficiency may
influence differently the formation and morphology of IF protein
aggregates in neurons depending on the levels of other IF proteins. The
absence of NF-L reduced the size of perikaryal swellings in transgenic
mice overproducing human NF-H proteins (Fig. 2), whereas it
precipitated the formation of IF aggregates and motor neuron death in
mice overexpressing peripherin (Beaulieu et al., 1999a). Distinct
neuronal cell populations may express various levels of each neuronal
IF protein and therefore may develop IF protein aggregates with
different characteristics in a situation of NF-L deficiency. Our
findings may be of importance for neurodegenerative diseases because
decreased levels of NF-L mRNA are associated with ALS (Bergeron et al.,
1994), Alzheimer's disease (Crapper McLachlan et al., 1988), and to a
lower extent, aging (Parhad et al., 1995).
| |
FOOTNOTES |
Received March 14, 2000; revised April 27, 2000; accepted April 29, 2000.
This research was supported by the Medical Research Council of Canada
(MRC), the Amyotrophic Lateral Sclerosis (ALS) Association (USA), and the ALS Society of Canada. J-M.B. is a recipient of studentships from the Natural Science and Engineering Research Council
of Canada, Les Fonds de la Recherche en Santé du Québec, and the McDonald Stewart Foundation. J-P.J. has an MRC Senior Scholarship. The technical assistance of Pascale Hince, Daniel Houle,
and Gaetan Gagnon is gratefully acknowledged.
Correspondence should be addressed to Dr. Jean-Pierre Julien, The
Montreal General Hospital Research Institute, 1650 Cedar Avenue,
Montreal, Quebec, Canada, H3G 1A4. E-mail: mdju{at}musica.mcgill.ca.
Dr. Jacomy's present address: Institut National de la Recherche
Scientifique-Institut Armand-Frappier, Laval, Quebec, Canada H7V 1B7.
| |
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ABSTRACT
INTRODUCTION
