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The Journal of Neuroscience, August 1, 2002, 22(15):6515-6525
Deregulation of cdk5, Hyperphosphorylation, and Cytoskeletal
Pathology in the Niemann-Pick Type C Murine Model
Bitao
Bu1, 2,
Jin
Li1,
Peter
Davies3, and
Inez
Vincent1
1 Department of Pathology, University of Washington,
Seattle, Washington 98195, 2 Department of Neurology,
Tongji Hospital, Huazhong University of Science and Technology, 430030 Wuhan, China, and 3 Departments of Neuroscience and
Pathology, Albert Einstein College of Medicine, New York, New York
10461
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ABSTRACT |
NPC-1 gene mutations cause Niemann-Pick type C
(NPC), a neurodegenerative storage disease resulting in premature death
in humans. Spontaneous mutation of the NPC-1 gene in
mice generates a similar phenotype, usually with death ensuing by 12 weeks of age. Both human and murine NPC are characterized
neuropathologically by ballooned neurons distended with lipid storage,
axonal spheroid formation, demyelination, and widespread neuronal loss.
To elucidate the biochemical mechanism underlying this neuropathology,
we have investigated the phosphorylation of neuronal cytoskeletal
proteins in the brains of npc-1 mice. A spectrum of
antibodies against phosphorylated epitopes in neurofilaments (NFs) and
MAP2 and tau were used in immunohistochemical and immunoblotting
analyses of 4- to 12-week-old mice. Multiple sites in NFs, MAP2, and
tau were hyperphosphorylated as early as 4 weeks of age and correlated with a significant increase in activity of the cyclin-dependent kinase
5 (cdk5) and accumulation of its more potent activator, p25, a
proteolytic fragment of p35. At 5 weeks of age, the development of
axonal spheroids was noted in the pons. p25 and cdk5 coaccumulated with
hyperphosphorylated cytoskeletal proteins in axon spheroids. These
various abnormalities escalated with each additional week of age,
spreading to other regions of the brainstem, basal ganglia, cerebellum,
and eventually, the cortex. Our data suggest that focal deregulation of
cdk5/p25 in axons leads to cytoskeletal abnormalities and eventual
neurodegeneration in NPC. The npc-1 mouse is a valuable
in vivo model for determining how and when cdk5 becomes
deregulated and whether cdk5 inhibitors would be useful in blocking NPC neurodegeneration.
Key words:
cdk5; p35; neurodegeneration; Niemann-Pick disease type
C; cholesterol; axon spheroid; lipid rafts; caveolas; neurofilament
phosphorylation; tau phosphorylation; cytoskeletal pathology
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INTRODUCTION |
Niemann-Pick type C disease (NPC)
is a rare, autosomal recessive, fatal, lysosomal lipidosis affecting
multiple organs (Vanier et al., 1991a ,b; Scriver et al., 2001). The
disease is caused predominantly by mutations in the NPC-1
gene and less frequently in the HE1 (also referred to as
NPC-2) gene (Naureckiene et al., 2000; Millat et al., 2001;
Scriver et al., 2001). The NPC-1 gene encodes for a
cholesterol transporter in late endosomes, and the HE1 gene
encodes for a lysosomal cholesterol-binding protein. Neuropathologically, NPC is characterized by neurons distended with
lipid storage material having a foamy appearance, dendritic and axonal
abnormalities, demyelination, and widespread neuronal loss (Elleder et
al., 1985; Love et al., 1995; Suzuki et al., 1995). In addition,
neurofibrillary tangles (NFTs), a diagnostic lesion of Alzheimer's
disease (AD), are also a consistent finding, particularly in cases with
a prolonged course of disease (Auer et al., 1995; Love et al., 1995;
Suzuki et al., 1995) (H. H. Klünemann, B. Bu, J. Husseman,
M. Elleder, K. Suzuki, S. Salamant, S. Love, H. Budka, C. Fligner, T. Bird, L.-W. Jin, D. Nochlin, and I. Vincent, unpublished observations).
How these various neuropathologic features result from altered
cholesterol metabolism in NPC is a mystery and a rather difficult one
to resolve given the rarity of the disease.
A tremendous asset for unraveling the neuropathologic effects of
NPC-1 mutations is the BALB/cNpc-1nih mouse, which harbors a
spontaneous mutation in its npc-1 gene (Loftus et al.,
1997). Mice with homozygous npc-1 mutations
(npc-1 mice) display extensive lipid storage accumulation,
neuroaxonal dystrophy, and neuronal loss, similar to that of human NPC
(Higashi et al., 1993; Suzuki et al., 1995; Sawamura et al., 2001).
Cholesterol (Xie et al., 1999; Sawamura et al., 2001) and
glycosphingolipids such as gangliosides GM2 and neutral
glycolipids (Walkley, 1995; Zervas et al., 2001) are the predominant
constituents of storage material in the npc-1 mouse brain.
Curiously, however, neither alleviation of cholesterol (Patterson et
al., 1993; Erickson et al., 2000; Camargo et al., 2001) nor ganglioside
storage (Liu et al., 2000) ameliorate the neurological phenotype or
progressive neuronal loss in npc-1 mice or feline NPC,
although lipid storage was effectively reduced in neurons and other
cells. Thus, it is yet unclear what mechanism underlies neuronal
dysfunction and loss of neurons in NPC. A notable difference between
the npc-1 mouse and human NPC is the absence of NFTs in the
mouse (German et al., 2001a; Sawamura et al., 2001). However, in light
of the conspicuous axonal abnormalities in human, murine, and feline
NPC (Elleder et al., 1985; Higashi et al., 1993; Ong et al., 2001), we
wondered whether cytoskeletal abnormalities contribute to neuronal
dysfunction and degeneration in NPC. Therefore, we have undertaken a
detailed characterization of cytoskeletal protein phosphorylation in
the brains of npc-1 mice.
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MATERIALS AND METHODS |
All procedures in this study were approved by the Internal
Review Board and Animal Use and Care Committee of the University of Washington.
Npc-1 mice. A breeding pair of heterozygous
npc-1 mice obtained from The Jackson Laboratory (Bar Harbor,
ME) was bred to generate wild-type (+/+), heterozygous
(npc-1 +/ ), and homozygous (npc-1 /) mice,
which were identified using an established PCR-based method (Loftus et
al., 1997). Tail biopsies for genotyping were performed at the time of
weaning (i.e., at ~3 weeks). Only / mice have been reported to
display pathology (Tanaka et al., 1988). In initial studies, we
screened +/ mice and confirmed the absence of cytoskeletal pathology
in this genotype. Hence, all further study concentrated on comparisons
of / mice with +/+ siblings. Twenty-eight npc-1 /
mice (4, 5, 7, and 9 weeks of age, n = 3; 6 and 8 weeks
of age, n = 5; 10, 11, and 12 weeks of age,
n = 2) and a minimum of two age-matched (for each
week), wild-type littermates were analyzed by immunohistochemistry and immunoblotting.
Brain tissue. Mice were killed by carbon dioxide exposure
followed by decapitation. The brains were removed quickly and divided sagittally into halves. The right halves were immersion fixed with 4%
paraformaldehyde/PBS for 1 week and then embedded in paraffin. Where
indicated, some mice were transcardially perfused with 4% paraformaldehyde/PBS, and the brain was then processed for paraffin embedding. The paraffin-embedded blocks were sectioned at 6 µm for
histological analyses. The left halves were frozen at 80°C for
biochemical study. In some cases, the forebrain, cerebellum, and
brainstem were isolated and frozen separately for regional analysis.
Frozen hippocampus from a clinically and neuropathologically confirmed
AD case was used in parallel with the mouse samples as a control for
specificity of NFT antibodies.
Antibodies. The primary antibodies used in this study are
summarized in Table 1.
Immunohistochemistry and immunofluorescent labeling.
Immunohistochemical staining was performed on paraffin-embedded
sections as described previously (Vincent et al., 1997, 1998) with
modifications. Immersion-fixed sections were incubated with 88% formic
acid for 7 min to enhance antigen recovery and washed three times for
15 min with Tris-buffered saline (in mM): 10 Tris-HCl, pH 7.4, and 150 NaCl, before the addition of primary
antibody. Biotinylated, isotype-specific secondary antibodies followed
by HRP-labeled streptavidin were used to detect primary
antibody-specific binding, visualized using diaminobenzidine (DAB,
brown). Sections were counterstained with hematoxylin
(blue-purple).
Immunofluorescent labeling was conducted similarly (Vincent et al.,
1998). For visualization of specific antibody binding, streptavidin-conjugated Cy-3 (red, 1:500) or Alexa Fluor 488 (green, 1:500) was used. Nuclei were counterstained in 1 µg/ml
4,6-diamino-2-phenyliodole (DAPI, blue) in distilled water for 30 sec
at room temperature. Light and fluorescent micrographs were collected
using a Nikon (Tokyo, Japan) Optiphot microscope connected to a
computerized SPOT CCD camera (Diagnostic Instruments, Inc., Sterling
Heights, MI).
Preparation of brain extracts. Frozen tissue was weighed and
homogenized with a polytron in 10 volumes of ice-cold lysis buffer [in
mM: 10 Tris-HCl, 150 NaCl, 20 NaF, 1 mM sodium vanadate, 2 ethylene glycol-bis
( -aminoethylether)-N,N,N',N'-tetraacetic acid, 0.5%
Triton X-100, and 0.1% SDS] and proteinase inhibitor cocktail
(P-8340; Sigma, St. Louis, MO). The homogenates were aliquoted and
stored at 80°C.
Immunoblotting analysis. Frozen aliquots were thawed and
centrifuged at 12,000 × g at 4°C for 5 min, and the
soluble fraction was used for immunoblotting. The protein content in
supernatants was determined using a Bio-Rad (Hercules, CA) protein
assay kit and a Microplate Reader (Molecular Devices, Sunnyvale, CA).
The supernatants were then subjected to SDS-gel electrophoresis and immunoblotting analyses (Vincent et al., 1997). For the analyses of
MAP2 and neurofilaments (NFs), 10 µg of protein was loaded per lane;
for tau and cyclin-dependent kinase 5 (cdk5) studies, 40 µg of
protein was loaded per lane. For heat-stable proteins, supernatants
containing 30 µg of protein were boiled for 10 min at 100°C and
then centrifuged. The supernatants containing the heat-soluble protein
were resolved and immunoblotted according to routine procedures.
Immunoprecipitation. Protein (100 µg) from lysates in
equal volume was immunoprecipitated with 2 µg of cdk5 polyclonal
antibody as described previously (Vincent et al., 1997). The
immunoprecipitates (IPs) were processed for immunoblotting (as above)
or kinase assay as described below.
Kinase assay. The cdk5 IPs were washed twice with lysis
buffer and once with kinase buffer (50 mM HEPES,
pH 7.0, 10 mM MgCl2, 1 mM dithiothreitol, and 10 µM cold ATP), resuspended in 20 µl of kinase
buffer containing 10 µg of histone H1 and 0.5 µCi of [ -32P]ATP, and incubated at room
temperature for 20 min. To stop the reaction, 5 µl of 5× sample
buffer was added, and the samples were boiled for 5 min at 95°C.
Samples were separated by SDS-PAGE. Resolved proteins in the gel were
visualized with Coomassie blue dye, and the gels were then dried and
exposed to film for autoradiography.
Densitometric analysis. ECL and autoradiographic films were
scanned, the appropriate bands were outlined, and their densities were
measured using NIH image software. Statistical differences were
determined with Student's t test using Microsoft Excel
(Seattle, WA)
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RESULTS |
Hyperphosphorylation and accumulation of neurofilament protein in
npc-1 / mouse brain
The immunohistochemical staining with SMI 31 antibody was
performed on immersion-fixed sagittal sections. Consistent with the
expected localization of phosphorylated NFs (Julien and Mushynski, 1982), SMI 31 primarily stained axon tracts throughout the brain of
wild-type (+/+) mice (Fig.
1A,C,E).
In npc-1 / mice, SMI 31 stained numerous spot-like
structures in the same regions through which axons typically course
(Fig.1B,D,F shown at higher magnification in
Fig.1G,H, red, I,
arrowhead). The abnormal spot-like structures were first
noted in the pons at 5 weeks of age; they were present throughout the
basal ganglia (Fig. 1B), brainstem (Fig.
1D), and white matter of the cerebellum (data not
shown) by 7-8 weeks of age but were rarely seen in the hippocampus
(Fig. 1F, arrows). By 11-12 weeks of age,
they were observed in small numbers in the cerebral cortex as well
(data not shown). Although we did not directly analyze neuronal loss in
this study, disruption of the continuity of the Purkinje neuron layer
in the cerebellum was conspicuous at 6 weeks of age, and Purkinje cells
were rarely present after 9-10 weeks of age (data not shown). To
further characterize the spot-like structures, immunofluorescent
labeling with SMI 31 (red) and nuclear counterstaining with DAPI (blue)
was performed. The abnormally enlarged SMI 31-positive structures were
devoid of nuclei (Fig. 1G). Occasional perikaryal staining
was recognized by the presence of a nucleus within a larger-sized spot
(Fig. 1H, arrow) relative to the more
abundant smaller spot-like structures. Thus, the SMI 31-positive
spot-like structures most likely represent the axonal spheroids
described previously in human, murine, and feline NPC (Elleder et al.,
1985; Higashi et al., 1993; Ong et al., 2001). These axonal spheroids
often displayed a translucent core resembling lipid deposit (Fig.
1I, arrowhead). In general, neuronal
somata swollen with lipid-like material were unstained (Fig.
1I, arrow). The SMI 32 antibody
recognizing a nonphosphorylated epitope in 200 kDa high-molecular
weight NF (NF-H) stained a few neuronal cell bodies in the brainstem
and small fibers throughout the brain of +/+ mice (Fig.
1J). In / mice, SMI 32 immunolabeled numerous
axonal spheroids (Fig. 1K, arrows) with a
regional distribution (shown for the pons only, Fig.
1K) that was similar to those stained by SMI 31.
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Figure 1.
NF abnormalities in npc-1 /
mice. Immersion-fixed, paraffin-embedded sagittal brain sections from
7-week-old +/+ (A, C, E, J) and / (B,
D, F, G-I, K) mice were immunolabeled with antibodies
SMI 31 (A-I) or SMI 32 (J,
K). For some sections, antibody binding was visualized
with DAB (brown) and hematoxylin counterstain for
highlight ing the nuclei of all cells (blue-purple)
or with Cy-3 (red) and DAPI counterstain for nuclei
(blue). In the +/+ mouse, SMI 31 positively stained
bundles of processes throughout the brain [shown for the basal ganglia
(A), brainstem (C), and
hippocampus (E)]. In sharp contrast, intense
spheroid-like structures were observed in vast numbers in the basal
ganglia (B) and brainstem
(D) and sparsely in the hippocampus
(F, arrows). The absence of nuclei within
the spheroidal structures (G) and their size
suggest that they are cross sections of swollen axons. Some rarer,
larger spots containing a nucleus resembled perikarya
(H, arrow in cortex). The gigantically
enlarged neurons containing storage were not stained with SMI 31 (I, arrow), in contrast to axonal
staining with a translucent core (I,
arrowhead). SMI 32 stained a few large neurons and small
fibers in the pons of the +/+ mouse (J) and
axonal spheroids principally in the brainstem of / mice
(K, arrows). Magnification:
A-F, 4×; G-K, 40×.
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To verify that the histological abnormalities were a result of
increased phosphorylation and/or accumulation of NFs, immunoblotting analyses were conducted with whole-brain lysates from +/+ and /
mice. Replicate blots were stained with SMI 31, SMI 32, and R39, a pan
NF antibody recognizing all of the NF isoforms. The intensities of the
NF-H, 160 kDa medium-molecular weight (NF-M), and 68 kDa
low-molecular weight (NF-L) NF bands stained with R39 from four
8-week-old npc-1 mice and four controls were quantitated densitometrically, and the results indicated that the levels of the
NF-H, NF-M, and NF-L were increased by 2.4-, 1.5-, and 1.17-fold (p < 0.03 for all), respectively, in the
npc-1 mice. These changes in NF levels were observed,
whereas the levels of many other proteins, such as tau, MAP2, cdk5
(shown in Figs. 4 and 5), and neuronal-specific nuclear protein (NeuN)
(Brazelton et al., 2000), in the same samples, were invariant. The
results for NeuN are shown in Figure 2
and indicate equal protein loading of all lanes. With respect to
phosphorylation, a sixfold increase in SMI 31 immunoreactivity was
observed with NF-M, but there was no significant increase in SMI31
immunoreactivity with NF-H (Fig. 2, SMI 31). These results suggest that
NF-M is the only hyperphosphorylated NF isoform npc-1 mouse
brain. They also imply that phosphorylation of NF-H did not increase in
proportion to the increase in total NF-H levels, which corresponds to a
net accumulation of nonphosphorylated NF-H in the diseased mice. This interpretation was supported by the marked increase in SMI 32 immunoreactivity with NF-H in the npc-2 mouse brain samples
(Fig. 2, SMI 32) and the increased SMI 32 immunoreactivity with axonal spheroids (Fig. 1K). Neither SMI 31 nor SMI 32 stained any bands corresponding to tau, as has been observed in AD
(Sternberger and Sternberger, 1983).
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Figure 2.
Increased levels of NF-H
and increased phosphorylation of NF-M in npc-1 /
mice. Whole-brain lysates containing 10 µg of protein from 8-week-old
+/+ and 4- and 8-week-old / mice were resolved on 8% gels,
transferred to nitrocellulose, and blotted with the indicated
antibodies. The pan NF antibody R39 identified the three major NF
isoforms (marked NF-H, NF-M, and NF-L, respectively). The
intensities of the bands visualized with SMI 31 and R39 were
quantitated, and the results indicated a significant increase in the
levels of all three isoforms and a marked increase in
phosphorylation of the NF-M isoform. Equivalent loading of all samples
is demonstrated with the NeuN antibody recognizing the 46-48 kDa
neuronal-specific antigens.
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Hyperphosphorylated tau and MAP2 accumulate in npc-1
mouse brain
To determine whether other cytoskeletal proteins normally
localized in axons and dendrites (i.e., tau and MAP2) (Binder et al.,
1986; Matus, 1990) are also modified, their phosphorylation status was
examined in npc-1 mice. A library of anti-tau
antibodies [i.e., paired helical filament-1 (PHF-1), CP-13, CP-22,
MC-6, MC-1, ALZ-50, and TG-5] raised against PHFs from
AD brains (Table 1) was used. Overall, the pattern of staining
obtained with the phosphotau antibodies was similar in that bundles of
fibers were stained in +/+ mice (Fig.
3A, PHF-1, D,
CP-22) and numerous axon spheroids were detected in / mice (Fig.
3B, PHF-1, E, CP-22). Classic NFTs were not
observed with any of the anti-tau antibodies. Some perikarya of neurons
(Fig. 3C, PHF-1) in the brainstem and basal ganglia was
visible. Tau-positive axon spheroids were seen in the brainstem, basal
ganglia, and white matter of / mice as early as 5 weeks of age and
became more conspicuous over the following 7 weeks (shown only in
8-week-old mice) (Fig. 3B,C, PHF-1, E, CP-22).
The pattern of staining with the CP-10 antibody recognizing
phosphothreonine-231 in tau was somewhat different from that of
the above phosphotau antibodies. CP-10 exhibited weak staining of
neuronal cell bodies in +/+ mice (Fig. 3F), but in
/ mice there was a dramatic increase in the staining of neurons both in the cytoplasm and in the nucleus (Fig. 3G,
arrow). Some neurons containing foamy material were
conspicuously unstained (Fig. 3G, *), but others were
stained (Fig. 3G, arrowhead). Curiously, the
conformation- and sequence-dependent tau antibodies ALZ-50, MC-1, and
TG-5 did not react with axonal spheroids or any other pathological
feature in the npc-1 mice (data not shown). These antibodies
stained axon fibers weakly in +/+ mice and fewer numbers of similar
fibers in npc-1 / mice (data not shown).
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Figure 3.
Tau and MAP2 abnormalities in the
npc-1 / mouse brain. Sagittal brain sections from
+/+ (A, D, F, H) and 8-week-old / mice
(B, C, E, G, I, J) were immunostained with PHF-1
(A-C), CP-22 (D, E), CP-10
(F, G), and AP-18 (H-J). PHF-1
stained bundles of processes in the brainstem and white matter of +/+
mice (A, pons) but numerous axonal spheroids
(B, arrows) and some perikarya
(C) of neurons in similar regions of the /
mouse brain. CP-22 displayed much weaker staining of processes in +/+
mice (D) and a similar pattern of axonal
spheroids (arrows) in the brainstem of / mice
(E, pons). CP-10 exhibited weak staining of neuronal
cell bodies in +/+ mice (F, pons), but in / mice
there was a dramatic increase in the staining of neurons both in the
cytoplasm and in the nucleus (G, large
arrow). Some neurons containing foamy material were
conspicuously unstained (G, asterisk),
but others were CP-10 positive (arrowhead). AP-18
was primarily negative in the +/+ mouse brain
(H) but labeled the soma and dendrites of
several neurons (arrows) in the thalamus
(I). J, One such neuron
with prominent staining of the soma and apical dendrite. Magnification:
A, B, D-G, I, J, 40×; C, H,
100×.
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The AP-18 antibody recognizing a phosphoepitope in MAP2 gave
positive results with perfusion-fixed sections only. In 7-week-old / mice, AP-18 displayed intense immunoreactivity in the
somatodendritic compartment of large neurons of the thalamus (Fig.
3I, arrows). The apical dendrite was prominently
labeled in many of these cells (Fig. 3J). Axonal
spheroids in the thalamus and brainstem were labeled to a lesser degree
with AP-18 (data not shown) than with tau or NF antibodies. The
antibody did not show any staining in +/+ brain sections (Fig.
3H). AP-20, an antibody recognizing total MAP2 (i.e.,
the 240-280 kDa MAP2a and MAP2b isoforms), positively labeled the soma
of many neurons throughout the brain, without any striking difference
between the two groups of mice (data not shown).
Immunoblotting analyses of the microtubule-associated proteins was
conducted with heat-stable supernatants from +/+ and / mice. An
increase in tau phosphorylation was detected in npc-1 /
mice at the earliest time point tested (i.e., 4 weeks of age) and
increased further through 10 weeks of age (only data from the 4- and
8-week-old mice are shown) (Fig. 4). In
4-week-old npc-1 / mice, an increase in intensity of
phosphotau antibody immunoreactivity with tau was seen compared with
+/+ mice, although there was no striking change in electrophoretic
mobility (Fig. 4, blots PHF-1, CP-13, and CP-10). In 8-week-old
npc-1 / mice, the triplet pattern of hyperphosphorylated
tau similar to that of AD became apparent with most of the antibodies
(Fig. 4, compare bands stained with CP-13 in 8-week-old
npc-1 / mice and AD). The average increase in
immunoreactivity with phosphotau antibodies in 8-week-old
npc-1 mice compared with age-matched +/+ mice was threefold
for CP-10 (n = 4) and PHF-1 (n = 8) and
twofold for CP-13 (n = 8). The tau sequence and
conformation antibodies TG-5, ALZ-50, and MC-1 showed no difference in
the intensity of the tau bands between the two groups of mice,
indicating similar levels of tau protein in both (Fig. 4, only TG-5 and
ALZ-50 shown). Together, these data support a net increase in the
phosphorylation of tau in npc-1 mice.
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Figure 4.
Increased phosphorylation of
microtubule-associated proteins in npc-1 / mice. Ten
micrograms of whole-brain, heat-stable protein from +/+ and / mice
and a typical AD case were blotted with the indicated antibodies
(supernatant only for AP-18). Ten and 8% gels were used for detection
of tau and MAP2, respectively. An increase in intensity and in the
apparent molecular weight of tau was observed with PHF-1 and CP-13 in
4-week-old and even more so in 8-week-old / mice, consistent with
hyperphosphorylation of tau. The pattern of tau bands at 8 weeks of age
resembled that of AD-tau. CP-10-detected phosphorylated tau was
increased threefold in / mice. CP-22 raised against and shown to
recognize phosphorylated tau did not react with tau in the BALB/c
strain of mouse. However, the antibody detected a molecule of ~180
kDa that was more highly phosphorylated in / mice. The tau sequence
antibodies TG-5 and ALZ-50 displayed equivalent amounts of tau in all
lanes. AP-18 immunoreactivity showed increased
phosphorylation of the 280 kDa MAP2a and MAP2b and 70 kDa MAP2c protein
/ mice, particularly at 4 weeks of age. AP-20 did not recognize
MAP2c but showed invariable amounts of total MAP2
(2a+2b) protein in all lanes.
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Unexpectedly, CP-22 and MC-6, which were raised against purified PHF,
did not react with tau in either +/+ or / mice. However, both
antibodies recognized a protein of ~180 kDa that was markedly elevated in heat-stable supernatants from npc-1 mice (Fig.
4, shown for CP-22 only). The size of this band is larger than the 97-110 kDa high-molecular weight tau species (Georgieff et al., 1991),
and we found that it was more abundant in the supernatant fraction
without any heat treatment, suggesting that it is primarily heat labile
(data not shown).
Consistent with the accumulation of phosphorylated MAP2 by
immunohistochemistry, immunoblotting demonstrated that the
phosphorylated high-molecular weight 240-280 kDa MAP2a and MAP2b
isoforms of and the low-molecular weight 70 kDa MAP2c isoform were
enriched in / mice, particularly at 4 weeks of age (Fig. 4, AP-18).
At 8 weeks of age, no increase in phosphorylation of MAP2a and MAP2b was detected, but MAP2c appeared to be hyperphosphorylated. A duplicate
blot stained with the AP-20 antibody recognizing the primary sequence
in MAP2a and MAP2b showed similar amounts of these proteins in /
mice relative to +/+ mice (Fig. 4, AP-20).
p25 levels and cdk5 activity are increased in
npc-1 / mouse brain
Previous evidence has suggested that the PHF-1, CP-13, SMI 31, and
AP-18 phosphoepitopes are generated by cdk5 kinase (Berling et al.,
1994; Patrick et al., 1999; Nguyen et al., 2001) that is enriched in
axons (Tsai et al., 1993). Therefore, we compared the levels of cdk5
and its activator, p35, and cdk5 activity in / and +/+ mice.
Immunoblotting analyses showed that the cdk5 levels were invariable in
/ mice compared with +/+ mice (Fig. 5A, cdk5,
mouse). p35 levels were also unchanged, but an elevation in p25, a
C-terminal truncated fragment of p35 (Patrick et al., 1999), was
detected in / mouse brains from 5 to 12 weeks of age (Fig.
5A, p35 mouse). A similar pattern of constant cdk5 levels but increased p25 levels is seen in human NPC (Fig. 5A,
human). Elevated p25 correlates with an increase in cdk5 activity,
because p25 has a longer half-life and is a better activator of cdk5
than p35 (Patrick et al., 1999). To see whether cdk5 activity is
altered in / mice, the kinase was immunoprecipitated from
whole-brain lysates and tested for its phosphorylation activity toward
histone H1 in vitro (Fig. 5B, cdk5 IP/kinase
assay). The recovery of cdk5 in all of the immunoprecipitates was
similar as judged by immunoblotting with a cdk5 monoclonal antibody
(Fig. 5B, whole brain, cdk5 blots in top row),
and Coomassie blue staining of the gel showed equivalent amounts of H1
substrate in the kinase reaction in all lanes (Fig. 5, whole brain, gel
shown in second row). However, incorporation of
labeled phosphate from [-32P]ATP into
H1 was significantly higher with cdk5 IPs from / mice than from +/+
mice (Fig. 5B, whole brain, H1 phosphorylation shown in
third row). The increase was most striking between 4 and 7 weeks of age and less so at  8 weeks of age. The lower increase in
cdk5 activity in older mice may be a result of neuronal loss, which at
8 weeks of age is most prominent in the cerebellum but becomes more
significant in other regions as well in later stages of the disease.
The average increase in cdk5 activity in whole-brain lysates from 5- to
10-week-old / mice was 1.6-fold (Fig. 5B, whole brain,
histogram; p < 0.01; n = 12).
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Figure 5.
cdk5 and p25 abnormalities in the
npc-1 / mouse brain. A, cdk5/p35
immunoblots. Whole-brain lysates containing 40 µg of protein from +/+
and / mice were resolved on 12% gels and blotted with the
indicated antibodies. cdk5 levels remained unchanged in / mice
compared with controls. The p35 C-terminal antibody showed no
difference in p35 levels but an enrichment of the 25 kDa p35 fragment
(p25) in / mice (left panel, mouse).
Similar preparations containing 100 µg of protein from a human
control and an NPC hippocampus were immunoblotted with cdk5 and p35C
antibodies. cdk5 and p35 levels were invariant, but p25 was elevated in
NPC (right panel, human).
B, cdk5 IP/kinase activity. Whole-brain lysates
(left panel) from +/+ and / mice were
immuno- precipitated with cdk5 (C-8) antibody. Immunoprecipitate
(cdk5 IP) was immunoblotted with a different cdk5 antibody to show that
the relative recoveries of enzyme in the samples were similar
(Cdk5). Catalytic aliquots of cdk5 immunoprecipitate
were incubated with kinase buffer containing histone H1 and
[-32P]-ATP for 20 min at 30°C, and the reaction
mixtures were resolved on 12% gels. The incorporation of
[-32P]ATP into histone H1 (H1) was
significantly higher with cdk5 IP from / mice compared with +/+
mice (left panel), on the basis of equivalent H1
loading demonstrated by staining of the gel with Coomassie blue
(gel). The cdk5 activity in 5- to
10-week-old / mice was increased 1.5-fold over that seen in +/+
mice (p < 0.01; n = 12;
left panel). Right panel, The
increase in cdk5 activity in the forebrain (Forebr.) and
brainstem (B.stem) but not in the cerebellum
(Cblm.) of a 5-week-old / mouse compared with those
of a 5-week-old +/+ mouse (all scales, ×1000). C,
P-GSK-3 immunoblots. Whole-brain lysates containing 50 µg of
protein from +/+ and / mice were resolved on 10% gels and blotted
with P-GSK-3 antibody (P-GSK-3) or primary
sequence-dependent antibody (GSK-3). The intensities of
the resulting bands were quantitated densitometrically, and no
significant differences (p = 0.11) were
observed between the npc-1 and control mice.
|
|
Given the regional distribution of axonal spheroids in npc-1
brain, we also performed a regional comparison of cdk5 activities. The
cerebellum, brainstem, and remaining forebrain from 5-week-old mice
were analyzed. As was done with cdk5 IPs from whole-brain lysates,
equivalent recovery of cdk5 in the IP and the equivalent amount of H1
were verified with the samples from different brain regions (Fig.
5B, Forebr., B.stem, Cblm., respectively, cdk5
blot shown in first row, H1 protein in gel shown in
second row). An increase in the amount of labeled phosphate
incorporated into H1 was visible in the forebrain but was far more
striking in the brainstem and barely noticeable in the cerebellum (Fig.
5B, Forebr., B.stem, Cblm., H1 phosphorylation
shown in third row). Quantitation of the data from two such
determinations using samples from two different sets of mice revealed a
sixfold increase in cdk5 activity in the brainstem and a twofold
increase in the forebrain but no change in the cerebellum (Fig.
5B, histogram).
To support the significance of these cdk5 activity changes in NPC,
another proline-directed kinase [i.e., glycogen synthase kinase-3 (GSK-3)] that has been shown to phosphorylate
sites similar to those of cdk5 in the above cytoskeletal proteins
(Julien and Mushynski, 1998; Mattson, 2001) was explored. The GSK-3
kinase is inactivated by phosphorylation of its ser9 residue (Grimes and Jope, 2001). The phospho-GSK-3 (P-GSK-3) (ser9) antibody recognizing this phosphoepitope provides a convenient means of detecting changes in the activity of the kinase by tracing the phosphorylation status of this site. This P-GSK-3 antibody and another primary sequence-dependent GSK-3 antibody were used to explore the phosphorylation status and levels of GSK-3 in the npc-1 mouse brain. Replicate blots were generated using
brain lysates from five 5- to 9-week-old npc-1 mice and
three controls of similar ages. One blot was stained with the
P-GSK-3 antibody and the other with the sequence antibody (GSK-3);
neither antibody revealed any consistent change in GSK-3
immunoreactivity in NPC (Fig. 5C). This experiment was run
in duplicate, and the intensities of the bands obtained with each
antibody in the npc-1 mice were compared with the
corresponding bands in control mice. There was no significant
difference (p = 0.11) in kinase levels or
activity in the npc-1 mice (Fig. 5C). Reprobing
the blots with NeuN antibody confirmed that all of the lanes were
equally loaded (data not shown).
p25 accumulates and colocalizes with hyperphosphorylated NFs in
axon spheroids of NPC / mouse brain
To further determine whether the cdk5 activity changes
were relevant to the cytoskeletal pathology in npc-1 mice,
immunohistochemical studies of cdk5 and p35 were conducted using
perfusion-fixed brain sections. Despite the constant cdk5 levels in the
immunoblots discussed above, cdk5 antibodies displayed an increase in
immunoreactivity in some deformed neurons (Fig.
6B, arrow)
and axon speroids (arrowhead) in the brainstem and basal
ganglia of / mice compared with +/+ mice of the same age (Fig.
6A). An N-terminal p35 antibody recognizing only p35
but not p25 stained bundles of fibers in +/+ mice and fewer numbers of
fibers in / mice but no axonal spheroids (data not shown). In
contrast, a C-terminal p35 antibody recognizing both p35 and p25
exhibited intense and extensive staining of axonal spheroids in the
brainstem, basal ganglia, and white matter of the cerebellum (Fig. 6,
only brainstem shown, F, green), but in +/+ mice,
its immunoreactivity was faint and localized in fibers and in
soma of a few neurons (arrows in Fig. 6C,
green). C-terminal p35 antibody-specific staining of NFTs
suggested the accumulation of p25 in NFTs of the AD brain (Patrick et
al., 1999). Thus, it appears that the C-terminal p35 antibody-specific
staining of axonal spheroids may also suggest that increased p25 is
localized to pathological structures in npc-1 mouse brain.
To gather additional support for this idea, double labeling with
C-terminal p35 antibody (green) and SMI 31 (red) was performed.
Wild-type sections did not show significant colocalization of
C-terminal p35 antibody immunoreactivity and SMI 31 (Fig.
6C,D, respectively, and merged image in
E). The micrograph shown from the brain of a 7-week-old npc-1 / mouse instead indicates considerable overlap in
localization of the two antigens in axonal spheroids
(arrows, Fig. 6, C-terminal p35 antibody, green,
F and H, SMI 31, red, G and
H) throughout the brain.
View larger version (135K):
[in this window]
[in a new window]
|
Figure 6.
p35 accumulates and colocalizes with
phosphorylated NF in axonal spheroids of npc-1 /
mice. Perfusion-fixed, paraffin-embedded sections from 7-week-old +/+
(A, C-E) and / (B, F-H) mice
were immunohistochemically labeled with cdk5 (brown) and
counterstained with hematoxylin (blue-purple) or
immunofluorescently labeled with p35 (Alexa 488, green)
and SMI 31 (Cy-3, red) and counterstained with DAPI
(blue). cdk5 barely stained neurons in +/+ mice
(A, pontine nucleus). In contrast, cdk5 immunoreactivity
was increased in the soma of some deformed neurons
(arrow) and in the axonal spheroids
(arrowhead) in the brainstem of / mice
(B, pontine nucleus). p35 faintly labeled the soma of
neurons in the brainstem of +/+ mice (arrows in
C and E, pons) and displayed little
colocalization with SMI 31 immunoreactivity (D and
merged image in E). In the / mouse
brain, intense p35 immunoreactivity was seen in the axonal spheroids
(F, pons), which perfectly matched the accumulation of
SMI 31 immunoreactivity (G and arrows in
merged image in H).
Inset in H, Gigantically enlarged axons
containing p35 (green) and SMI 31 (red) immunoreactivity in the cerebral cortex.
Magnification: A, B, 100×; C-H,
40×.
|
|
| |
DISCUSSION |
The cardinal findings of this study include elevated levels of
NF-H and hyperphosphorylation of NF-M, tau, and MAP2 in the npc-1 mouse brain. These changes coincide with deregulation
of the neuronal cdk5/p35 kinase complex. Accelerated proteolytic cleavage of p35 to p25, a more powerful cdk5 activator, correlates with
increased cdk5 activity, and p25 colocalizes with hyperphosphorylated cytoskeletal proteins in the most conspicuous neuropathological lesion,
the axonal spheroid. These focally enlarged axonal abnormalities are
concentrated in the brainstem, basal ganglia, and white matter of the
cerebellum, regions containing long myelinated axons and having high
NPC-1 gene expression levels (Prasad et al., 2000). Their
abundance is directly proportional to the increase in cdk5 activity in
each brain area. Chronologically, cdk5 activation and
hyperphosphorylation are dramatic at 4 weeks of age, the earliest time
point studied here, and axonal spheroids were first detected in the
pons at 5 weeks of age. The severity of these changes escalated rapidly, reaching a maximum at 8-9 weeks of age, after which point marked brain atrophy became obvious. All of the biochemical changes detected here are observed in human NPC (Klünemann, Bu, Husseman, Elleder, Suzuki, Salamant, Love, Budka, Fligner, Bird, Jin, Nochlin, and Vincent, unpublished observations), but it is difficult to evaluate
their temporal characteristics using autopsy brain tissue. The sequence
of neuropathological events that we have delineated in the
npc-1 mouse highlights an opportunity to inhibit the NPC neurodegenerative cascade with cdk5 inhibitors. Our studies also emphasize the value of the npc-1 mouse model for testing and
developing this therapeutic strategy and unraveling additional details
of NPC neuropathogenesis.
NFTs were not detected in the npc-1 mouse brain using tau
antibodies or Bielchowsky silver reagent (data not shown), confirming previous studies that used thioflavin S (German et al., 2001a) and in
contrast to the widespread occurrence of NFTs in human NPC (Auer et
al., 1995; Suzuki et al., 1995). Curiously, even with a threefold
increase in the phosphorylation of tau at some sites, the
npc-1 mouse brain was devoid of TG-5, ALZ-50, and MC-1 immunoreactivity. Such sequence- and conformation-dependent tau antibodies rarely stain neurons of normal rodent or human brain; their
epitopes are sensitive to fixation, but they react strongly with AD
neurons because their fixation sensitivity is alleviated by
phosphorylation and aggregation (Pollock and Wood, 1988; Papasozomenos, 1989; Weaver et al., 2000). Our data would argue that
hyperphosphorylation of tau at sites 202 (CP-13) and 396-404 (PHF-1)
in axons and of CP-10-positive tau in neuronal soma are inadequate for
eliciting TG-5, ALZ-50, and MC-1 immunoreactivity in either neuronal
compartment. Perhaps phosphorylation at additional sites or other
modifications (Gonzalez et al., 1998; Takeda et al., 2000) is required
to expose these epitopes in situ. The absence of such
additional modifications in the npc-1 mouse brain might also
be the key to their inability to form NFTs. The CP-22 and MC-6
antibodies recognize phosphothr-175 and phosphoser-235 in
lysine-serine/threonine-proline motifs, respectively, in AD
PHF (P. Davies, unpublished observations). Neither antibody
reacted with tau in npc-1 mice, suggesting that these sites
are not phosphorylated in NPC. Instead, both antibodies recognized a
180 kDa heat labile protein present in axon spheroids. The possibility
of this protein being a tau aggregate is excluded by its negative
staining with other tau antibodies. It could be a degradation product
of a larger protein containing similar proline-directed phosphorylation
sites (i.e., NF-H or MAP2). All three MAP2 isoforms are
hyperphosphorylated in the neuronal somatodendritic compartment of
npc-1 mice, but additional experiments are required to
clarify their relationship with the 180 kDa protein.
Regarding the individual NF isoforms, the increase in NF-H levels was
greater than the increase in SMI 31 immunoreactivity with NF-H; the
increase in NF-M levels was less than the increased SMI 31 immunoreactivity with NF-M. These data translate into a selective
increase in expression or reduced phosphorylation or degradation of
NF-H and a selective increase in NF-M phosphorylation in NPC. The
robust staining of axon spheroids with SMI 31 and SMI 32 suggests that
a mixture of nonphosphorylated and hyperphosphorylated NF protein
accumulates in the lesions. These results confirm earlier electron
microscopic evidence for intermediate filaments in spheroids from human
NPC (Elleder et al., 1985). However, axons normally contain
hyperphosphorylated NFs, whereas perikarya contain hypophosphorylated NFs (Pant and Veeranna, 1995). Many neurodegenerative diseases, such as
AD (Schmidt et al., 1991; Nixon, 1993), Parkinson's disease (Forno et
al., 1986), and amyotrophic lateral sclerosis (ALS) (Julien, 1995), are
characterized by perikaryal accumulations of hyperphosphorylated NFs.
Axonal dilations have been documented in ALS and experimental models of
axotomy (Stone et al., 2001) and in transgenic mice overexpressing four
repeat human tau (Spittaels et al., 1999). These conditions are also
associated with perikaryal NF or tau accumulation, implying disruption
of proximal axonal transport. The absence of somatic accumulation of
hyperphosphorylated tau and NFs in npc-1 mice suggests a
defect downstream of the proximal axon in NPC. Using silver staining
(Patel et al., 1999) and Golgi impregnation (Zervas et al., 2001), it
was noted that terminal fields of axons and dendrites are the earliest
sites of degeneration in npc-1 mice. Together, these data
suggest that NPC neurodegeneration proceeds along a distal-to-proximal
axis in long axons. The integrity of these axons has been thought to depend on the cycling of cholesterol between surrounding glial cells
and the axons themselves (Xie et al., 1999), which may explain their special vulnerability to the lipid disturbances caused by NPC-1 mutations.
In accordance with this more "distal" origin of NPC axonopathy is
the enrichment of cdk5 in distal axonal segments (Nikolic et al.,
1996). cdk5 normally mediates phosphorylation of NFs, tau, and MAP2 and
their detachment from microtubules in axons (Matsushita et al., 1996;
Wada et al., 1998). In npc-1 mice, elevated p25
levels appear to be the trigger for cdk5 activation in distal axons,
because p25 was enriched in cdk5 IPs from npc-1 brain and colocalized with hyperphosphorylated NFs in axonal spheroids. Moreover,
the levels and activity of the GSK-3 kinase, another enzyme that has
been implicated in NF and tau phosphorylation, were unaltered in the
npc-1 mice. In transgenic mice overexpressing p25, a twofold
increase in cdk5 activity resulted in tau and NF hyperphosphorylation
and cytoskeletal pathology (Ahlijanian et al., 2000). Similarly,
increased p25 activity and a twofold increase in cdk5 activity
correlated with tau and NF hyperphosphorylation and motor neuron
degeneration in the SODG37R ALS mouse model (Nguyen et al., 2001). In
npc-1 mice, we measured as much as a sixfold increase in
p25-associated cdk5 activity in the brainstem, where hyperphosphorylation and cytoskeletal abnormalities were intense. A
causal role for cdk5 in NPC is also supported by evidence linking its
activity to phosphorylation of serines 202 and 396-404 in tau, the SMI
31 epitope in NF, and the AP-18 epitope in MAP2 (Berling et al., 1994;
Patrick et al., 1999; Ahlijanian et al., 2000; Grant et al., 2001;
Nguyen et al., 2001). Presently, it is not possible to exclude the
involvement of other kinases in NPC. Sawamura et al. (2001) reported
upregulation of MAP kinase, and we (Bu, Klünemann, Suzuki,
Husseman, Bird, Jin, and Vincent, unpublished observations) have
observed aberrant expression of cell division cycle kinase (cdc2) and cyclin B1 in the npc-1 mouse cerebellum.
Even with equivalent recovery of cdk5 in cdk5 IPs from the cerebellum
and other brain regions, cdk5 activity and p25 were not elevated in the
cerebellum, suggesting that other kinases mediate hyperphosphorylation in this region (Sawamura et al., 2001). The cerebellum has low p35
expression but is rich in p39 (Tang et al., 1995), which is degraded to
a more effective activator, p29 (Patzke et al., 2002). We did not
explore p39/p29 here, but if p29 was elevated in the npc-1
cerebellum, an increase in cdk5 activity would have been detected using
cdk5 IPs. p35 expression is induced by MAP kinase (Harada et al.,
2001), which may explain the sustained p35 levels in npc-1
mice that were observed even with its increased conversion to p25. The
thr-231 tau epitope recognized by the CP-10 antibody when
phosphorylated is a better acceptor site for cdc2 than cdk5. CP-10
immunoreactivity is produced abundantly in npc-1 mice and is
the only tau phosphoepitope that we detected in neuronal soma. Thus,
although cdk5 may be the principal effector of axonal tau and NF in
NPC, a somatic pool of tau may be modified by a different proline-directed kinase.
Cholesterol, glycosphingolipids, and
glycosyl-phosphatidylinositol-anchored proteins cluster in
distinct cell membrane microdomains, called lipid rafts, and
raft-enriched membrane invaginations called caveolas (Kurzchalia and
Parton, 1999) constitute "signal transduction centers,"
coordinating extracellular signals with neuronal function and stability
(Brown and London, 1998; Masserini et al., 1999). cdk5 mediates such a
link in axons (Maccioni et al., 2001), and it is intriguing that p35
through its interaction with Rac GTPase (Nikolic et al., 1998);
calpain, the protease that converts p35 to cytosolic p25
(Kulkarni et al., 1999; Bialkowska et al., 2000; Kusakawa et al., 2000;
Lee et al., 2000); and a fraction of cellular NPC-1 (Garver
et al., 2000) are all present in caveolas. We propose that
caveolas play a crucial role in NPC neuropathology. Alterations of the
caveolar scaffold protein caveolin-1 and of annexin II in
npc-1 mice (Garver et al., 1997a,b) support this idea. The demyelination induced by NPC-1 mutations (Elleder et al.,
1985; German et al., 2002) may potentiate axonal pathology, because myelin regulates the levels and activity of calpain (Chakrabarti et
al., 1990; Persson and Karlsson, 1991), the expression and phosphorylation of NFs (Starr et al., 1996; Gotow et al., 1999), and
the density and stability of axonal microtubules (Sanchez et al., 2000;
Kirkpatrick et al., 2001). It should be possible to unravel the
temporal and spatial relationships of these events in the
npc-1 mouse model.
| |
FOOTNOTES |
Received Feb. 21, 2002; revised May 10, 2002; accepted May 22, 2002.
This work was supported by a grant from the Seattle Jim Lambright
Medical Research Foundation (I.V.) and Grants AG12721 (I.V.), P50 AG
05136-16 (Alzheimer's Disease Research Center, M.R.), and MH38623 (P.D.) from the National Institute on Aging. We thank Drs.
Harish C. Pant and Lester Binder for their gifts of antibodies and Drs.
Kinuko Suzuki and Steven Walkely for providing us with npc-1 mouse brain for preliminary studies. We also thank
Dr. Jan Hallows for her reading of this manuscript and her suggestions.
Correspondence should be addressed to Inez Vincent, University of
Washington, Department of Pathology, K072 HSB, Box 357705, 1959 Northeast Pacific Avenue, Seattle, WA 98195. E-mail:
ivincent{at}u.washington.edu.
| |
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ABSTRACT
INTRODUCTION
