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The Journal of Neuroscience, April 15, 1999, 19(8):2974-2986
Overexpression of  -Internexin Causes Abnormal Neurofilamentous
Accumulations and Motor Coordination Deficits in Transgenic Mice
Gee Y.
Ching1,
Chung-Liang
Chien2,
Roberto
Flores1, and
Ronald K. H.
Liem1
1 Departments of Pathology and Anatomy and Cell
Biology, Columbia University College of Physicians and Surgeons, New
York, New York 10032, and 2 Department of Anatomy, National
Taiwan University School of Medicine, Taipei, Taiwan 100, Republic of
China
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ABSTRACT |
 -Internexin is the first neuronal intermediate filament (IF)
protein expressed in postmitotic neurons of the developing nervous system. In the adult, its expression is restricted to mature neurons in
the CNS. To study the potential role of -internexin in
neurodegeneration, we have generated transgenic mice that overexpress
rat -internexin. The total levels of -internexin expressed in the
hemizygous and homozygous transgenic mice were ~2 and ~3 times the
normal level, respectively. Overexpression of -internexin resulted
in the formation of cerebellar torpedoes as early as 1 month of age.
These torpedoes are abnormal swellings of Purkinje cell axons that are
usually seen in neurodegenerative diseases involving the cerebellum. EM studies showed accumulations of high levels of IFs and abnormal organelles in the torpedoes and soma of Purkinje cells, as well as in
the large pyramidal neurons of the neocortex and in the ventral
anterior and posteromedial nuclei of the thalamus. Behavioral tests
demonstrate that these mice have a deficit in motor coordination as
early as 3 months of age, consistent with the morphological neuronal
changes. Our data further demonstrate that the neurofilamentous inclusions also lead to progressive loss of neurons in the aged transgenic mice. The motor coordination deficit and the loss of neurons
are transgene dosage-dependent. These data yield direct evidence that
high levels of misaccumulated neuronal IFs lead to neuronal
dysfunction, progressive neurodegeneration, and ultimate loss of
neurons. Moreover, the degrees of neuronal dysfunction and degeneration
are proportional to the levels of misaccumulated neuronal IFs.
Key words:
-internexin; neurofilament; intermediate filament; neurodegeneration; cytoskeleton; transgenic mice; motor deficits
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INTRODUCTION |
-Internexin is a neuronal
intermediate filament (IF) protein that is expressed earlier and more
abundantly than the neurofilament triplet proteins (NFTPs) throughout
the developing CNS (for review, see Fliegner and Liem, 1991 ). As
development continues, the levels of -internexin decrease, whereas
those of the NFTPs increase (Kaplan et al., 1990; Fliegner et al.,
1994). In adult CNS, -internexin is colocalized with the NFTPs in
most axons, but is present at lower levels in large neurons and is
found in cerebellar granule cells, which lack the NFTPs (Kaplan et al.,
1990). -Internexin can self-assemble and coassemble with each of the
NFTPs into filaments, whereas the NFTPs are obligate heteropolymers
(Ching and Liem, 1993, 1998; Lee et al., 1993). These properties
suggest that -internexin may play a role in stabilizing
small-diameter axons and act as a scaffold on which the NFTPs
coassemble during development.
Abnormal accumulations of neuronal IFs are pathological hallmarks of
many neurodegenerative diseases, such as amyotrophic lateral sclerosis
(ALS), Lewy body-type dementias, and Parkinson's disease (Munoz et
al., 1988; Sasaki et al., 1989; Galloway et al., 1992; Trojanowski and
Lee, 1994). Recent transgenic mouse models have shown that
overexpression of human NF-H (Cote et al., 1993) or mouse NF-L (Xu et
al., 1993) results in axonal swellings and perikaryal accumulation of
neuronal IFs in motor neurons, as well as skeletal muscle atrophy. The
mice develop signs of muscle weakness and reduced kinetic activity,
reminiscent of those found in ALS. Transgenic mice expressing a point
mutant of NF-L show neuronal abnormalities resulting in motor neuron
death and skeletal muscle atrophy (Lee et al., 1994). NF-H mutations
have also been found in sporadic ALS patients (Figlewicz et al.,
1994).
Overexpression of wild-type or mutant NFTPs does not always result in
ALS-like phenotypes. Transgenic mice that express a NF-H/ -galactosidase fusion protein show perikaryal accumulations of
IFs in their motor neurons but do not develop motor neuron degeneration
(Eyer and Peterson, 1994). The animals begin to show tremors and
selective Purkinje cell loss only at 1 year of age (Tu et al., 1997).
Their Purkinje cells contain Lewy body-like inclusions. Transgenic mice
that express wild-type human NF-M have normal motor neurons in the
spinal cord and exhibit perikaryal accumulations of neurofilaments in
other neurons at 12, but not 3, months of age (Vickers et al., 1994).
Surprisingly, transgenic mice that overexpress wild-type mouse NF-H or
NF-M show neither muscle atrophy nor motor neuron loss, despite
prominent axonal swellings and perikaryal neurofilamentous
accumulations in motor neurons (Wong et al., 1995; Marszalek et al.,
1996).
Thus, overexpressions of different NFTPs have differential effects on
neurons. Because -internexin differs from the NFTPs in assembly
properties and expression pattern, its overexpression may cause a
different type of neuropathy and provide additional insights into
mechanisms of neuronal dysfunction and neurodegeneration. Our present
study shows that subpopulations of CNS neurons appear to be relatively
more vulnerable to filamentous misaccumulations induced by
-internexin overexpression. Moreover, the transgenic mice show a
transgene dosage-dependent deficit in motor coordination.
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MATERIALS AND METHODS |
Generation of transgenic mice. To obtain a
full-length rat -internexin genomic clone, a 6.4 kb
BamHI-XhoI fragment and a 14.5 kb
XhoI fragment, isolated from the rat -internexin genomic clones  G-1 and G-2 (Ching and Liem, 1991), respectively,
were ligated to the BamHI-SalI digested
DashII phage vector (Stratagene, La Jolla, CA). The resulting
genomic clone was designated D-1. The transgene construct
pSP72N-ES16K was cloned by ligating the 13 kb EcoRI
fragment from D-1 and the 3.5 kb
EcoRI-SalI fragment from G-2 together with
the EcoRI-SalI digested pSP72N plasmid vector,
which was modified from pSP72 (Promega, Madison, WI) by changing the
EcoRV cloning site to a NotI site. To obtain the transgene fragment for generation of transgenic mice, the 16.5 kb
NotI-SalI fragment from pSP72N-ES16K,
consisting of the entire rat -internexin gene with 1.2 kb of its 5'
flanking sequence and 3.5 kb of its 3' flanking sequence, was isolated
from agarose gels and purified by glass powder. The resulting 16.5 kb
transgene fragment was further purified by Sephadex G50 spin columns
(Boehringer Mannheim, Indianapolis, IN) and prepared at 3 µg/ml in
the 10 mM Tris-HCl, pH 7.4, 10 mM NaCl, and 0.2 mM EDTA buffer. Transgenic mouse founders were generated by
microinjecting the transgene fragment into fertilized eggs isolated
from B6CBA F1/J mice (The Jackson Laboratory, Bar
Harbor, ME) according to the protocol of Hogan et al. (1986). Two
transgenic mouse lines 16K-T5 and 16K-T21 were established.
Hemizygous offspring were obtained by breeding the transgenic mice with
B6CBA F1/J, and homozygous offspring were obtained
by breeding the transgenic mice within each line.
To determine the presence of the transgene in the mice, small tail
pieces were cut from the mice, and DNA was extracted from these tails
as described (Hogan et al., 1986). Tail DNA was digested with
BamHI, separated on 0.8% agarose gels, and transferred to Zeta-Probe GT membranes (Bio-Rad, Hercules, CA). The 4.4 kb
BamHI fragment from G-2, consisting of the third exon
and 3' flanking sequence of the rat -internexin gene, was used as a
hybridization probe to detect the rat but not the mouse -internexin
gene. To determine which transgenic mice were homozygous with respect
to the transgene, equal amounts of tail DNA from the hemizygous and homozygous mice were used for Southern blot transfer, and a twice stronger rat -internexin hybridization signal on the autoradiogram indicated the homozygosity of the mice. To check the accuracy of the
quantities of tail DNA on Southern blots, the membranes were
rehybridized with a probe for the endogenous cdk5 gene (Sun et al.,
1996), which served as an internal reference. The homozygosity was
further confirmed by breeding the mice.
Production of mouse monoclonal antibodies. A 0.8 kb
BglII-BamHI fragment encoding the
C-terminal amino acid residues 340-505 of rat -internexin
and consisting of the SV40 poly A signal was isolated from pRSV-
(Ching and Liem, 1993), blunt-ended with Klenow, and attached to
BamHI linkers (CGCGGATCCGCG). After BamHI digestion, the resulting 0.8 kb fragment was cloned into the
BamHI site on the pET16b vector (Novagen, Madison, WI). The
clone, designated BB, was transformed into the bacterial strain
BL21(DE3) for expression. Expression of the -internexin C-terminal
peptide, which also contains an N-terminal histidine tag encoded by the
pET16b vector, was induced by 0.5 mM
isopropyl--D-thiogalactopyranoside for 4 hr.
Bacterial cell pellets were suspended in binding buffer (5 mM imidazole, 0.5 mM NaCl, and 20 mM Tris-HCl, pH 7.9) and sonicated, and the cell lysate was
centrifuged at 20,000 × g for 15 min at 4°C. The
resulting pellet was resuspended and incubated in binding buffer
containing 6 M urea on ice for 1 hr. The cell extract was
centrifuged at 39,000 × g for 20 min at 4°C. The
supernatant was filtered through a 0.45 µm membrane and then purified
by column chromatography using His·Bind metal chelation resins under
denaturing conditions according to the manufacturer's protocol
(Novagen). Protein fractions containing the purified -internexin
peptide were dialyzed extensively against 10 mM Tris-HCl,
pH 7.5, at 4°C. The precipitated protein obtained after dialysis was
solubilized by boiling briefly in 10 mM Tris-HCl, pH 7.5, containing 1% SDS. For immunization, the purified antigen (100 µg/immunization) was mixed with 0.2 ml saline containing 25 µg of
monophosphoryl lipid A plus synthetic trehalose dicorynomycolate
emulsion (Ribi Immunochem Research), an adjuvant, and injected
subcutaneously in two sites on Balb/c mice. Booster injections were
given every 3 weeks. Two weeks after the last antigen/adjuvant
injection, another boost was given without adjuvant. Three days later,
spleen cells were removed from the immunized mice for fusion with NS-1
myeloma cells, and monoclonal antibodies were produced according to the
method of Kohler and Milstein (1976). After immunoblotting and
immunohistochemical screening, two monoclonal antibodies, mAb12 and
mAb3G10, were isolated. mAb12 recognizes only rat
-internexin, and mAb3G10 reacts with both mouse and rat
-internexin.
Antibodies. The following antibodies were obtained
commercially (Sigma, St. Louis, MO): mouse monoclonal antibodies to
NF-L, NF-M, and NF-H (clones NR4, NN18, and N52, respectively); SMI31 and SMI36, which detect the phosphorylated epitopes on NF-M and NF-H;
SMI32, which recognizes the dephosphorylated forms of NF-M and NF-H;
and tau-2, which detects both phosphorylated and dephosphorylated forms
of tau. Rabbit polyclonal antibody to calbindin 28 kDa was purchased
(Swant, Bellinzona, Switzerland). Rabbit polyclonal antibody to GFAP
was previously described (Wang et al., 1984).
Western blot analysis. Mouse tissues were homogenized in 10 mM Tris-HCl, pH 7.5, and 1% SDS, boiled for 5 min, and
then centrifuged at 13,000 × g for 5 min to remove
insoluble materials. The resulting supernatant contained total proteins
extracted from the tissues. Protein concentrations were determined by
Bradford assay (Bradford, 1976). Equal amounts of proteins were
electrophoresed in 8 or 10% polyacrylamide-SDS gels (Laemmli, 1970)
and then electrotransferred to nitrocellulose filters (Towbin et al.,
1979). The filters containing proteins were incubated in PBS containing
5% bovine serum albumin for 30 min, washed with PBS, and incubated in
PBS containing primary antibodies for 1 hr. After several washes, they
were incubated in PBS containing horseradish peroxidase-conjugated
secondary antibodies for 30 min. They were subsequently washed and
treated with enhanced chemiluminescence (ECL) reagents (Amersham,
Arlington Heights, IL) for 1 min and exposed to x-ray films. Several
exposures of an autoradiogram were used for densitometric analysis.
Immunocytochemistry. Mice were anesthetized and perfused
with PBS containing 4% paraformaldehyde. Frozen cryostat sections of
10-15 µm thickness were prepared from the fixed tissues. The tissue
sections were incubated in ice-cold methanol for 10 min, washed, and
incubated in PBS containing 5% normal goat serum for 30 min. They were
washed with PBS and then incubated in PBS containing primary antibody
for 1 hr. After several washes, they were incubated in PBS containing
horseradish peroxidase-conjugated secondary antibody for 30 min. They
were then washed and treated with PBS containing 0.012% hydrogen
peroxide and 0.5 µg/µl diaminobenzidine (DAB). The tissue slides
were mounted with Permount (Fisher Scientific, Houston, TX). The
DAB/peroxidase-immunostaining procedure was modified for some primary
antibodies: methanol incubation was omitted, 0.1% Triton X-100 was
added to PBS, and tissues were incubated with primary antibody
overnight at 4°C. Indirect immunofluorescence staining using
rhodamine- or fluorescent-conjugated secondary antibodies was performed
as previously described (Ching and Liem, 1993).
Electron microscopy. Mice were anesthetized and perfused
with 4% paraformaldehyde and 2% glutaraldehyde in 0.1 M
cacodylate buffer, pH 7.4. Tissue blocks were immersed in the same
fixative for 24 hr at 4°C, rinsed in cacodylate buffer, and
post-fixed in 1% osmium tetroxide for 2 hr. After three washes with
cacodylate buffer, each sample was dehydrated in a graded series of
ethanol and embedded in Epon-Araldite resin. Plastic sections (1 µm)
were stained with toluidine blue and examined for light microscopy. Ultrathin sections were stained with uranyl acetate and lead citrate and examined with a Hitachi H-7100 electron microscope.
Behavioral tests. Hemizygous, homozygous, and nontransgenic
mice of 3 and 6 months of age were tested between 1:00 and 5:00 P.M. in
all behavioral experiments. The genotypes of the mice were unknown to
the observer at the time of the tests. The apparatus were cleaned
thoroughly after each mouse was tested. Statistical significance in
performance differences between the genotype groups were calculated
using the F test.
The open-field test was used to measure the overall locomotor
activities and motor posture patterns (Gerlai et al., 1993). The
open-field apparatus was a large opaque cage (12 × 28 cm) whose
bottom was covered with a 4 × 4 cm square grid. Mice were habituated to the open-field apparatus twice: 1 hr on the day before
testing and 1 hr on the test day. Individual mice were placed in the
center of the cage, and their movements were quantified for 20 min. The
following behaviors were recorded: locomotion score (number of squares
crossed on the floor grid), frequencies (number of times in a test
period) of rearing and defecation, and duration (as a percentage of the
test period) of grooming.
The rotorod test was used to measure motor coordination and balance
(Janicke et al., 1983; Luo et al., 1996). The rotorod apparatus,
Rotarod MECO, was purchased from Columbus Instruments and consists of a
1.75 inch rod with a rubber outer texture for grip. The rotorod speed
was electronically gauged. The mice were habituated on the rotorod
apparatus before testing by placing them on the rod for 1 min at the
rotating speed of 2 revolutions per minute (rpm). The habituation was
repeated after the mice were given a 1 min rest. After habituation, the
mice were given a 5 min rest and tested at a rotating speed of 10 rpm.
Timing started when the mice were placed on the rotating rod and
stopped when the mice fell off from the rotating rod or after 3 min
elapsed, whichever came first. After a 3 min rest, the test was
repeated. The retention times on the rotorod were averaged for each mouse.
To compare the performance between the 4.5-month-old hemizygous mice
and their nontransgenic littermates on the rotorod, the mice were
tested at the rotating speed of 17 rpm as described above, except that
timing stopped when the mice fell off from the rotating rod or after 4 min elapsed, whichever came first.
Quantitations of Purkinje cells. For each transgenic mouse
genotype examined at 12 and 18 months of age, three or four pairs of
transgenic/nontransgenic mice per age group were used for quantitation of Purkinje cells present in cerebellum. A complete set of
10-µm-thick paraffin-embedded brain sections was prepared from each
mouse, and every fifteenth section was picked and stained with cresyl violet. Comparable sections from the transgenic mice and their nontransgenic littermates were selected for cell counting. The presence
of cell nucleus was used as a criterion for scoring the number of
Purkinje cells present within each selected section. For each mouse
pair, the total number of Purkinje cells from the set of the transgenic
mouse sections was calculated as a percentage of that of the
nontransgenic littermate. The final value of the Purkinje cell number
determined for each transgenic mouse genotype group at an age point was
the average of these calculated percentages obtained from quantitation
of three or four pairs of transgenic/nontransgenic mice.
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RESULTS |
Neuron-specific overexpression of -internexin in
transgenic mice
We previously isolated two genomic clones containing partial
sequences of the rat -internexin gene (Ching and Liem, 1991). To
generate transgenic mice overexpressing -internexin, we used the two
genomic clones to construct a transgene, which consists of the entire
rat -internexin gene with 1.2 kb of 5' flanking sequence and 3.5 kb
of 3' flanking sequence. The resulting 16.5 kb transgene fragment was
microinjected into the pronuclei of fertilized mouse eggs. Two
transgenic mouse lines, 16K-T5 and 16K-T21, were established.
These two lines expressed and transmitted the transgene in a Mendelian
manner. To distinguish the expression of the transgenic product, rat
-internexin, from that of the endogenous mouse protein, we prepared
a monoclonal antibody that is specific for the rat protein. Rat and
mouse -internexin share high homology in their primary sequence but
differ in a short stretch of amino acids at their C-terminus (Chien and
Liem, 1994). We therefore used a bacterially produced peptide
consisting of the C-terminal region of rat -internexin as an
immunogen and generated a number of monoclonal antibodies. One of these
monoclonal antibodies, mAb12, recognizes rat but not mouse
-internexin. Another monoclonal antibody, mAb3G10, recognizes
both rat and mouse -internexin (see Figs.
1 and 2 for
antibody specificity).
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Figure 1.
Western blot analysis of -internexin and NFTPs
in transgenic mice. A, Total cellular proteins extracted
from the brains (with cerebella removed) (Br), cerebella
(Cb), and spinal cords (S) of
3-month-old 16K-T5 and 16K-T21 hemizygous mice and nontransgenic
(NT) littermates were separated by
SDS-polyacrylamide gel electrophoresis and electrotransferred to
nitrocellulose filters. The filters were immunostained with
anti--internexin antibodies mAb12 (a) or
mAb3G10 (b); monoclonal antibodies to NF-L
(c), NF-M (d), or NF-H
(e). Several different exposures of the
autoradiograms were used for densitometric analysis: rat and
mouse -internexin were expressed at approximately the same
levels, except that the level of rat -internexin in the Br fraction
of 16K-T5 is ~1.2 times that of mouse -internexin; the
endogenous levels of NFTPs remain unaltered. B, Western
blots containing total cellular proteins extracted from the brains
(with cerebella removed) (Br), cerebella
(Cb), and spinal cords (S) of
3-month-old 16K-T5 and 16K-T21 homozygous mice and nontransgenic
(NT) littermates were immunostained with
anti--internexin antibody mAb3G10. Densitometric analysis showed
that rat -internexin was expressed at ~2 times the level of mouse
-internexin in the homozygotes, except that the level of rat
-internexin is ~2.5 times that of mouse -internexin in the Br
fraction of 16K-T5.
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Figure 2.
Indirect immunofluorescence staining of cerebella
from 4.5-month-old transgenic and nontransgenic mice with
anti--internexin antibodies. Sagittal sections (15 µm) of
cerebella from 16k-T5 (A) and 16k-T21
(B) hemizygous mice and nontransgenic littermates
(C, D) were stained with
anti--internexin antibodies. A and C
were stained with mAb3G10, which detects both mouse and rat
-internexin. B and D were stained with
mAb12, which specifically recognizes rat -internexin. Perikaryal
immunostaining of Purkinje cells and torpedoes were detected in the
transgenic mice, but not in their nontransgenic littermates.
p, Purkinje cell; t, torpedo;
m, molecular layer; g, granular layer.
Scale bar, 45 µm.
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Western blot and densitometric analyses showed that wild-type rat
-internexin was expressed in the brains (with cerebella removed),
cerebella, and spinal cords of the 3-month-old hemizygous mice from
16K-T5 and 16K-T21 at approximately the same levels as the
endogenous mouse -internexin (Fig. 1A). This
twofold increase in the total levels of -internexin had no effect on
the expression levels of the NFTPs in the transgenic mice (Fig.
1A). Immunoblotting and immunocytochemical analyses
performed on various tissues such as brain, spinal cord, heart, lung,
liver, spleen, kidney, stomach, intestines, and muscle showed that
expression of rat -internexin in the transgenic mice was restricted
to neurons. Immunocytochemical examination of the transgenic
mouse neural tissues also revealed that rat -internexin was present
in the same regions where the endogenous mouse -internexin is
expressed. Furthermore, the expression patterns of the transgene in the
two transgenic mouse lines were found to be identical.
To increase the levels of -internexin further, we bred the
hemizygous mice within each transgenic mouse line to obtain transgenic mice that are homozygous with respect to the transgene. Western blot
and densitometric analyses showed that the total -internexin levels
increase to approximately threefold in the homozygous mice as expected
(Fig. 1B), whereas the levels of NFTPs remain
unaltered (data not shown).
Abnormal accumulation of neuronal intermediate filaments and
formation of cerebellar torpedoes
Immunocytochemical analyses showed that both the hemizygous and
homozygous mice of 16K-T5 and 16K-T21 contain abnormally intense
anti--internexin antibody staining in the perikarya of specific
groups of neurons in the cerebellum, neocortex, and thalamus. The
abnormal immunostaining patterns, seen as early as 1 month of age,
appeared similar between the hemizygous and homozygous mice, except
that the neuronal perikarya were more strongly stained by the
-internexin antibody in the homozygotes. Furthermore, ultrastructural studies showed that there was a greater accumulation of
intermediate filaments in the neuronal perikarya of the homozygous mice
than in those of the hemizygous mice. Immunocytochemical and
ultrastructural analyses revealed that the two mouse lines have similar
morphological changes in the CNS (also see below).
In the cerebella of 4.5-month-old hemizygous and homozygous mice of
16K-T5 and 16K-T21 (Fig.
2A,B), the perikarya of Purkinje cells were heavily stained by the anti--internexin antibodies mAb12, which specifically recognizes rat -internexin, and
mAb3G10, which reacts with both rat and mouse -internexin.
Immunostaining of the cerebellar white matter indicated colocalization
of rat and mouse -internexin within the nerve fibers. Numerous
fusiform swellings of the proximal portions of Purkinje cell axons
labeled by the antibodies to -internexin (Fig.
2A,B) were found in the granular
layer. These fusiform swellings, called torpedoes, are usually seen in
neurodegenerative diseases involving the cerebellum and in normal aging
(Duchen, 1984; Hirano, 1988). These torpedoes were also labeled by an
antibody to calbindin, which serves as a protein marker for Purkinje
cells (data not shown). The torpedoes were observed in the hemizygous
and homozygous mice at as early as 1 month of age. In the nontransgenic
littermates, Purkinje cell perikarya were not stained by the
anti--internexin antibody, and no torpedoes were observed (Fig.
2C). The torpedoes and perikarya of Purkinje cells of the
transgenic mice were also stained by the antibodies to the NFTPs (Fig.
5E,F). This result is
consistent with the ability of -internexin to coassemble with the
NFTPs into filaments (Ching and Liem, 1993, 1998) and the
colocalization of -internexin with the NFTPs in the CNS axons
(Kaplan et al., 1990). Thus, the immunocytochemical data showed that
overexpression of -internexin in the transgenic mice caused the
formation of torpedoes and abnormal accumulation of neuronal
intermediate filaments (composed of -internexin and NFTPs) in the
perikarya of Purkinje cells.
Ultrastructural analysis of the abnormal Purkinje cells by electron
microscopy revealed a swirl of closely packed, randomly oriented
neuronal intermediate filaments in the cytoplasm (Fig. 3A). The nucleus was
eccentrically localized, and many cellular organelles were pushed to
the periphery by the masses of neuronal intermediate filaments (data
not shown). Some of the mitochondria that were trapped within the
neurofilamentous masses looked abnormal or smaller than normal and
often associated with the smooth endoplasmic reticulum (Fig.
3A). The bulk of the torpedo consists of masses of closely
packed, randomly oriented neuronal intermediate filaments and
aggregates of organelles (Fig.
4A,B).
Some mitochondria and smooth endoplasmic reticulum looked abnormal, and
their membranes appeared to be continuous with each other. These
features are typical of torpedoes found in human neurodegenerative
diseases (Mann et al., 1980). The Purkinje cell axon distal to the
Purkinje cell soma and torpedo contains high levels of neuronal
intermediate filaments (Fig. 4C). Despite the perikaryal
accumulation of neuronal intermediate filaments and the formation of
torpedoes, no significant loss of neurons was observed at 4.5 months of
age.
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Figure 3.
Ultrastructural analysis of Purkinje cells and
neocortical pyramidal neurons from 4.5-month-old transgenic mice.
A, Ultrathin sections of cerebellum from an 16k-T5
homozygous mouse examined by electron microscopy revealed accumulation
of high levels of intermediate filaments in the soma of a Purkinje
cell. Lying within the neurofilamentous masses are aggregates of
mitochondria and sER, some of which appear to associate with each other
(arrows). Although the external membranes of these
mitochondria appear to be continuous with sER, some other mitochondria
look normal. B, Electron micrograph showed massive
accumulation of intermediate filaments in the soma of a neocortical
pyramidal neuron from a 4.5-month-old 16k-T5 homozygous mouse. Note
that the nucleus is eccentrically localized, and many cellular
organelles such as mitochondria are peripherally displaced. Some
mitochondria trapped within the masses of neuronal intermediate
filaments often aggregate and appear abnormal. The neuronal
intermediate filaments in A and B are
highly packed and randomly oriented. m, Mitochondria;
sER, smooth endoplasmic reticulum; n,
nucleus. Scale bars: A, 0.75 µm; B, 2.0 µm.
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Figure 4.
Ultrastructural analysis of cerebellar torpedo in
transgenic mice. A, Electron micrograph of the
cerebellum from a 4.5-month-old 16k-T21 homozygous mouse showed the
presence of a torpedo (arrow) in the granular layer.
B, High-magnification micrograph of the torpedo
(A, squared area) revealed the presence
of closely packed, randomly oriented neuronal intermediate filaments
and aggregates of organelles. C, High-magnification
micrograph of the Purkinje cell axon (A, squared
area) distal to the torpedo revealed the presence of high
levels of neuronal intermediate filaments. Scale bars:
A, 4.0 µm; B, 0.4 µm;
C, 0.5 µm.
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Immunocytochemical examination of the neocortex of 4.5-month-old
hemizygous and homozygous mice of the two transgenic lines also showed
the presence of filamentous inclusions in the perikarya of large
pyramidal neurons in layers III and V that were intensely stained by
the -internexin antibodies, mAb12 and mAb3G10 (Fig. 5A), as well as by the
antibodies to the NFTPs (Fig. 5C). Although these
filamentous inclusions have a neurofibrillary tangle-like appearance,
they were not stained by an anti-tau antibody that recognizes both the
phosphorylated and nonphosphorylated forms of tau (data not shown).
Because tau is the major constituent of the paired helical filaments
(Kondo et al., 1988; Goedert et al., 1989; Brion et al., 1991), the
absence of tau immunostaining indicated that the filamentous inclusions
in the perikarya of these neurons differ from the neurofibrillary
tangles of Alzheimer's disease. Similar perikaryal staining was also
detected in the ventral anterior nucleus and ventral posteromedial
nucleus of the transgenic mouse thalamus (Fig. 5B).
Examination of these abnormal neurons in the neocortex and thalamus by
electron microscopy revealed the presence of massive levels of closely
packed, randomly oriented neuronal intermediate filaments in the
cytoplasm and an eccentrically displaced nucleus (Fig. 3B).
Cellular organelles such as some of the mitochondria and smooth
endoplasmic reticulum that were trapped within the neuronal
intermediate filaments looked abnormal and often aggregated, but those
that were squeezed to the periphery by the neuronal intermediate
filaments appeared normal (Fig. 3B). In contrast to
the Purkinje cells, prominent swellings of proximal axons were not seen
in these neurons although higher neurofilament density was found in the
axons. Furthermore, no obvious neuronal loss was observed.
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Figure 5.
Immunocytochemical analysis of brains from
4.5-month-old transgenic mice. Sagittal sections of neocortex
(A, C, D), thalamus
(B), and cerebellum (E,
F) from 16k-T5 homozygous mice were stained
with antibodies mAb3G10, which reacts with both rat and mouse
-internexin (A, B), SMI32, which
detects only the nonphosphorylated forms of NF-M and NF-H
(C, E), or SMI36, which recognizes
phosphorylated epitopes on NF-M and NF-H (D,
F). Perikaryal immunostainings were observed with
mAb3G10 and SMI32, but not with SMI36. In contrast, torpedoes were
stained by both SMI32 and SMI36. p, Purkinje cell;
t, torpedo. Scale bar, 35 µm.
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|
Abnormal phosphorylation of NF-M and NF-H in perikaryal
neurofilamentous inclusions is often a hallmark of human
neurodegenerative diseases (Schmidt et al., 1987; Kato and Hirano,
1990; Nakazato et al., 1990; Sobue et al., 1990). To determine whether
NF-H and NF-M in the perikarya of the cerebellar Purkinje cells,
neocortical pyramidal neurons, and thalamic neurons described above
were phosphorylated, three monoclonal antibodies, SMI31, SMI32, and
SMI36, were used for immunocytochemical staining. SMI31 and SMI36
detect phosphorylated epitopes, whereas SMI32 recognizes only
nonphosphorylated forms of NF-M and NF-H. The results showed that the
perikarya of the neurons containing neurofilamentous inclusions were
stained by SMI32, but not by SMI31 and SMI36, indicating that the NF-M
and NF-H accumulated in the neuronal perikarya were not phosphorylated (Fig. 5C-F). In contrast, the cerebellar
torpedoes were stained by all three antibodies, demonstrating the
presence of both phosphorylated and nonphosphorylated forms of NF-M and
NF-H in the torpedoes (Fig.
5E,F).
Examination of the spinal cords of the transgenic mice did not reveal
any perikaryal accumulation of neuronal intermediate filaments in motor
neurons or axonal swellings, although expression of the transgene
increases the levels of -internexin in the spinal cord to ~3 times
the normal levels in the homozygotes. Consistent with the normal
morphology observed in the spinal cord, no skeletal muscle atrophy was
found in the transgenic mice (data not shown).
Deficit in motor coordination of transgenic mice
Neither the hemizygous nor homozygous mice of the
16K-T5 and 16K-T21 lines showed tremors or any other overt
symptoms of disorders. They also appeared to reproduce normally,
similar to their nontransgenic littermates. To assess any possible
deficits in motor performance of the transgenic mice resulting from the observed neuronal pathology, we subjected the mice to a battery of
behavioral tests. Two age groups (3 and 6 months) of homozygous, hemizygous, and nontransgenic mice were tested, and their performances were compared. The mice within each age group were weight-matched. Because the two transgenic mouse lines showed identical expression patterns, neuronal pathologies and motor performances in preliminary behavioral tests, they were pooled into the same groups for behavioral analysis.
Analysis by the open-field test revealed no significant differences
(p > 0.05, F test) in locomotion,
grooming, and rearing between the transgenic and nontransgenic mice
(Table 1). Although the frequency of
defecation was found significantly smaller in the 3-month-old
homozygous transgenic mice (p < 0.05) compared with that of the nontransgenic mice, it was not significant
(p > 0.05) among the 6-month-old mice. Taken
together, these results indicate that the overall locomotor activity
and gross motor-posture patterns of the transgenic mice were
indistinguishable from those of the nontransgenic mice.
The mice were subsequently tested for motor coordination and balance on
a rotorod. The results showed significant differences (p < 0.001, F test) in the retention
times on the rotorod between genotype groups (Fig.
6). The 3- and 6-month-old homozygous
mice performed more poorly than the age-matched hemizygous and
nontransgenic groups. There were no significant differences
(p > 0.05) in performance between the
hemizygous and nontransgenic mice at a rotating speed of 10 rpm (Fig.
6A). To determine whether the
-internexin-overexpressing hemizygous mice show impairment in motor
coordination on a rotorod at a higher speed, new groups of
4.5-month-old hemizygous and nontransgenic mice were tested at a
rotating speed of 17 rpm. At this speed, the retention times of the
hemizygous mice were much shorter than those of the age-matched
nontransgenic mice (Fig. 6B). These results suggest
that both the hemizygous and homozygous mice of 16K-T5 and
16K-T21 are impaired in the ability to perform a complex pattern of
movements involving high demands in motor coordination and balance.
Furthermore, the poorer performance of the homozygous mice, compared
with the hemizygous mice, indicates that the motor impairment is
transgene dosage-dependent.
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Figure 6.
Comparison of performance in rotorod test between
transgenic and nontransgenic mice. A,
-Internexin-overexpressing hemizygous (HZ) and
homozygous (HOM) mice of 3 and 6 months of age
and their age-matched nontransgenic (NT)
littermates (n = 10 each group) were tested, and
their retention times (in seconds) on the rotorod at a speed of 10 rpm
were compared. The -internexin-overexpressing homozygous mice, but
not the hemizygous mice, performed poorly compared with their
nontransgenic littermates (3-month-old mice,
F(2,27) = 56.7, p < 0.001; 6-month-old mice, F(2,27) = 27.5, p < 0.001. B,
-Internexin-overexpressing hemizygous (HZ) mice of
4.5 months of age and their age-matched nontransgenic
(NT) littermates (n = 15 each
group) were tested at a speed of 17 rpm on the rotorod, and their
retention times (in seconds) were compared. There is a significant
difference (p < 0.001; F
test) in the retention times between the hemizygous mice and their
nontransgenic littermates.
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Neuronal degeneration in transgenic mice
Examination of the brains from the older hemizygous and homozygous
mice (12-18 months of age) of 16K-T5 and 16K-T21 at the light
and electron microscopic levels revealed neuronal degeneration that is
progressive with increasing age of the mice (Figs.
7, 8, Table
2). Loss of neurons was seen in the
neocortex, thalamus, and cerebellum of aged transgenic mice (Fig. 7).
Ultrastructural analysis of the brains from 13-month-old transgenic
mice showed degenerating and degenerated processes in the molecular
layer (Fig. 8A) as well as degenerating torpedoes and
degenerated axons in the granular layer (Fig. 8B).
Degenerated and degenerating axons and processes were also found in the
neocortex (Fig. 8C) and the thalamus (data not shown). In
all three brain regions, microglia were often detected at the neuronal
degeneration sites (Fig. 8C). High levels of neuronal
intermediate filaments were still present in the perikarya of Purkinje
cells, neocortical pyramidal neurons, and thalamic neurons. In
contrast, degenerated processes and axons were very rarely found in the
nontransgenic littermates. These data showed that misaccumulation of
high levels of neuronal intermediate filaments ultimately led to
neuronal degeneration in the older transgenic mice.
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Figure 7.
Neuronal degeneration in aged transgenic mice.
Sagittal paraffin-embedded sections (10 µm) were stained with cresyl
violet and examined by light microscopy.
A-D, Examination of the cerebella from
12-month-old nontransgenic (A) and homozygous
(B) mice, and 18-month-old nontransgenic
(C) and homozygous (D) mice
revealed progressive loss of Purkinje cells as the transgenic mice aged
(C, D). Arrowhead in B
shows a Purkinje cell containing neurofilamentous inclusion.
m, Molecular layer; p, Purkinje cell;
g, granular layers. E, F,
Examination of the thalamus from 18-month-old nontransgenic
(E) and homozygous mice (F)
showed the presence of degenerated neurons (arrow) and
neurons containing neurofilamentous inclusions
(arrowhead) in the transgenic mice. Scale bars:
A-D, 50 µm; E,
F, 50 µm.
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Figure 8.
Ultrastructural analysis of the cerebellum and
neocortex from 13-month-old transgenic mice. Electron micrographs of
the cerebellum (A, B) and neocortex
(C) from 13-month-old 16k-T5 homozygous mice
revealed degenerated processes (arrows) in the molecular
layer (A) and a degenerating torpedo
(arrow) in the granular layer (B)
of the cerebellum, and degenerated processes (arrows)
and nearby microglial cells in the neocortex (C).
Scale bars: A, 2.0 µm; B, 1.5 µm;
C, 1.5 µm.
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Examination of the cerebella from the hemizygous and homozygous
transgenic mice at 12-18 months of age revealed progressive loss of
Purkinje cells (Fig. 7A-D). Quantitative
analysis of Purkinje cell numbers showed that at 12 months of age, the
hemizygous and homozygous mice had a decrease of 16.6 and 37.4%,
respectively, in the Purkinje cell population (Table 2). By 18 months of age, there is a further decrease in the number of Purkinje
cells, with a 60.6 and 71.5% loss in the hemizygous and homozygous
mice, respectively. Overall, the extent of Purkinje cell loss was
greater in the homozygous mice than in the hemizygous mice. Taken
together, these data demonstrated that misaccumulation of high levels
of neuronal intermediate filaments in Purkinje cells ultimately led to
the degeneration and death of these neurons.
| |
DISCUSSION |
Our studies demonstrate that overexpression of -internexin
induces the formation of cerebellar torpedoes and abnormal
accumulations of neuronal intermediate filaments (composed of
-internexin and the NFTPs) in the brains of the 16K-T5 and
16K-T21 hemizygous and homozygous mice as early as 1 month of age.
Furthermore, these mice exhibit a transgene dosage-dependent deficit in
motor coordination as early as 3 months of age. Because there is no
obvious loss of neurons at this age, the observation of a deficit in
the motor coordination of the transgenic mice indicates that the
functions of the affected neurons are compromised because of
misaccumulations of high levels of neuronal intermediate filaments. The
mechanism by which the accumulations of neuronal intermediate filaments occur in these animals is not clear. It is possible that the system responsible for transporting neuronal intermediate filaments to the
axons is overloaded by excessive amounts of -internexin in the
transgenic mice and as a result, -internexin as well as the NFTPs
accumulate in the neuronal perikarya and swollen axons.
Unlike some of the transgenic mice that express wild-type or mutant
NFTPs (Cote et al., 1993; Xu et al., 1993; Eyer and Peterson, 1994; Lee
et al., 1994; Wong et al., 1995; Marszalek et al., 1996), the 16K-T5
and 16K-T21 transgenic mice did not show any axonal swellings and
perikaryal accumulation of neuronal intermediate filaments in the motor
neurons of the spinal cord, although the expression levels of
-internexin increased in these neurons. Consistent with this result,
they also did not develop any skeletal muscle atrophy or ALS-like
phenotype. Because -internexin is normally expressed at much lower
levels than the NFTPs in the motor neurons of the spinal cord (Chiu et
al., 1989; Kaplan et al., 1990; Fliegner et al., 1994), the increased
levels of -internexin may still be insufficient to cause
misaccumulation of neuronal intermediate filaments in motor neurons.
In contrast, morphological analyses of the brains of the 16K-T5 and
16K-T21 mice revealed different groups of neurons that are
susceptible to the overexpression of -internexin. Abnormal perikaryal accumulations of neuronal intermediate filaments were detected in the Purkinje cells of the cerebellum, the large pyramidal neurons in layers III and V of the neocortex, and the ventral anterior
nucleus and ventral posteromedial nucleus of the thalamus. Prominent
swellings of the Purkinje cell axons also occurred, resulting in the
presence of numerous torpedoes in the granular layer of the cerebellum.
Thus, Purkinje cells appear to be the most affected by the
overexpression of -internexin. Although these abnormal morphological
changes were detected as early as 1 month of age, prominent loss of the
affected neurons was seen mainly in the aged ( 12 months) transgenic
mice, indicating a remarkable degree of tolerance of the neurons for
massive misaccumulation of neuronal intermediate filaments. This
neuronal tolerance may contribute in part to the slow process of
neuronal degeneration and hence the gradual progression of
neurodegenerative diseases with neurofilamentous inclusions.
Consistent with the slow process of neuronal degeneration in
neurodegenerative diseases, the neurodegeneration and ultimate loss of
neurons observed in the old -internexin-overexpressing transgenic
mice (12-18 months of age) are progressive with increasing mouse age.
By 18 months of age, prominent loss of Purkinje cells (60%) was
observed in both hemizygous and homozygous mice. Furthermore, the
extent of neuron loss is proportional to the levels of misaccumulated neuronal intermediate filaments, as indicated by the greater loss of
Purkinje cells in the homozygous mice compared with the hemizygous mice. Thus, our data yield direct evidence that misaccumulation of high
levels of neuronal intermediate filaments arising from overexpression
of -internexin can cause progressive neurodegeneration and neuron death.
The transgene dosage-dependent motor coordination deficits observed in
the -internexin-overexpressing transgenic mice demonstrate that high
levels of misaccumulated neuronal intermediate filaments can compromise
the normal functions of neurons containing filamentous inclusions. The
degree of this neuronal dysfunction is proportional to the levels of
misaccumulated neuronal intermediate filaments, as shown by the poorer
performance of the homozygous mice, compared with the hemizygous mice,
in the rotorod test. Moreover, this neuronal dysfunction occurs before
the neuronal degeneration and loss seen in the transgenic mice. The
resulting deficit in the motor coordination of the transgenic mice is
consistent with the observed morphological neuronal changes. Motor
coordination requires the normal operation of a number of neural
structures, which also include the motor cortex, thalamus, and
cerebellum (Thach et al., 1992; Llinas and Welsh, 1993; Ivry, 1997).
These neural structures together control and regulate movement
specification and initiation. The cerebellum, in particular, is widely
believed to play a crucial role in coordinating and modulating
unskilled and skilled movements and is implicated in regulating the
temporal patterns of movement. It also directs its output to the
cerebral cortex and thalamus. Purkinje cells, the neurons most affected
by the overexpression of -internexin, send their axons to the deep
cerebellar nuclei, the major output pathway from the cerebellum. The
abnormal pyramidal neurons containing neurofilamentous inclusions are
located in the premotor and motor areas of the neocortex. Thus, it is
not surprising that the transgenic mice with the morphological neuronal changes in the cerebellum, neocortex, and thalamus are impaired in
motor coordination.
Misaccumulation of high levels of neuronal intermediate filaments may
induce neuronal dysfunction and degeneration by at least two
mechanisms. The findings of some abnormal cellular organelles (such as
the mitochondria and smooth endoplasmic reticulum) entrapped within the
perikaryal neurofilamentous inclusions and an increase in the number of
these abnormal organelles in the older -internexin-overexpressing transgenic mice suggest that the cellular metabolism may be perturbed because of the organelle damages and a shortage of normal organelles required for neuronal function and viability, ultimately resulting in neuronal dysfunction and degeneration. Moreover, the
neuronal intermediate filaments present in the cerebellar torpedoes
and perikarya of the affected neurons in the
-internexin-overexpressing transgenic mice are randomly
oriented. Excessive accumulation of these disorganized neurofilamentous
cytoskeletons may cause defects in axonal transport of neurofilaments
and other cellular components essential for axonal maintenance. It has
previously been shown that overexpression of human NF-H in transgenic
mice impairs the axonal transport of NFTPs and cellular organelles (Collard et al., 1995). Retardation in the slow axonal transport of
cytoskeletal elements during aging (McQuarrie et al., 1989) may further
compound the impairment of axonal transport caused by the abnormal
accumulation of neuronal intermediate filaments, thereby accelerating
axonal degeneration in older transgenic mice.
The presence of phosphorylated NF-M and NF-H in perikaryal
neurofilamentous inclusions is often a pathological hallmark of many
neurodegenerative diseases (Schmidt et al., 1987; Kato and Hirano,
1990; Nakazato et al., 1990; Sobue et al., 1990). This abnormal
presence of phosphorylated NF-M and NF-H in neuronal perikarya has also
been observed in some of the transgenic mice overexpressing wild-type
or mutant NFTPs (Xu et al., 1993; Eyer and Peterson, 1994; Lee et al.,
1994; Tu et al., 1997). Study of neurofilamentous aggregates induced in
cultured neurons suggests that perikaryal accumulation of neuronal
intermediate filaments precedes the aberrant phosphorylation of NF-H in
the cell body (Straube-West et al., 1996). In contrast, phosphorylated
NF-M and NF-H were not detected in the perikarya of the neurons
containing the neurofilamentous inclusions in the
-internexin-overexpressing transgenic mice, but were found in the
axons where they are normally confined (Sternberger and Sternberger,
1983; Carden et al., 1987). It seems that the massive misaccumulation
of neuronal intermediate filaments caused by the overexpression of
-internexin does not induce any inappropriate activation of kinases
or inactivation of phosphatases in the affected neurons that could lead
to the aberrant phosphorylation of NF-M and NF-H in the perikarya.
In contrast to the NFTPs, little is known about the potential
involvement of -internexin in the pathogenesis of neurodegenerative diseases. The -internexin-overexpressing transgenic mice differ from
the NFTP-overexpressing transgenic mice in neuronal pathologies, as
well as in the phenotype induced by misaccumulation of massive neuronal
intermediate filaments, and hence provide an additional system for
elucidating mechanisms of neuronal dysfunction and degeneration. The
data obtained from the studies of the -internexin-overexpressing transgenic mice yield direct evidence that high levels of
misaccumulated neuronal intermediate filaments lead to neuronal
dysfunction, progressive neurodegeneration, and ultimate loss of
neurons. The use of both hemizygous and homozygous mice allows
demonstration of a direct correlation of the levels of misaccumulated
neuronal intermediate filaments to the degree of neuronal dysfunction
and degeneration observed. Furthermore, these transgenic mice show neuronal dysfunction preceding neuronal loss, a pattern characteristic of some of the human neurodegenerative diseases containing
neurofilamentous inclusions. In view of these data, the transgenic
mouse model presented here also provides some initial insights into the
role that -internexin may play in the pathogenesis of human
neurodegenerative diseases.
| |
FOOTNOTES |
Received Nov. 9, 1998; revised Jan. 28, 1999; accepted Jan. 28, 1999.
This work was supported by Grant NS15182 from the National Institutes
of Health. C.-L. Chien was supported by Grant NSC 88-2314-B-002-119 from National Science Council, Taiwan. We thank Dr. Frank Costantini and Ms. Xiao-lin Liang for generation of transgenic mouse founders, Dr.
Mary Bach for helpful advice on the behavioral experiments, Ms. Beth
Rosen and Mr. Reilly Coch for their excellent technical assistance, Mr.
Adam Nguyen and Ms. Allie Lui for assistance in DNA extraction and
tissue sectioning, and Drs. Dongming Sun and Conrad Leung for their
help in photographic printing.
Correspondence should be addressed to Dr. Ronald Liem, Department of
Pathology, Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, NY 10032.
| |
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TOP
ABSTRACT
INTRODUCTION
