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The Journal of Neuroscience, March 15, 1998, 18(6):2063-2074
Deficiency in Protein L-Isoaspartyl Methyltransferase
Results in a Fatal Progressive Epilepsy
Akihiro
Yamamoto1, 2,
Hideyuki
Takagi1,
Daisuke
Kitamura3,
Hozumi
Tatsuoka4,
Hirotake
Nakano5,
Hitoshi
Kawano6,
Hidehito
Kuroyanagi1,
Yu-ichi
Yahagi1,
Shin-ichiro
Kobayashi1,
Ken-ichi
Koizumi1,
Tsuyoshi
Sakai1,
Ken-ichi
Saito7,
Tanemichi
Chiba4,
Koki
Kawamura6,
Katsushi
Suzuki7,
Takeshi
Watanabe8,
Hiroshi
Mori2, and
Takuji
Shirasawa1, 9
1 Department of Neurophysiology, Tokyo Metropolitan
Institute of Gerontology, Tokyo-173, Japan, 2 Department of
Molecular Biology, Tokyo Institute for Psychiatry, Tokyo-156, Japan,
3 Research Institute for Biological Science, Science
University of Tokyo, Chiba-278, Japan, 4 Department of
Anatomy, Chiba University, Chiba-260, Japan, 5 Department
of Neurosurgery, National Center of Neurology and Psychiatry,
Tokyo-187, Japan, 6 Department of Anatomy, Keio University,
Tokyo-160, Japan, 7 Department of Veterinary Physiology,
Nippon Veterinary and Animal Science University, Tokyo-180, Japan,
8 Department of Molecular Immunology, Kyushu University,
Fukuoka-812, Japan, and 9 CREST, Japan Science and
Technology Corporation, Japan
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ABSTRACT |
Protein L-isoaspartyl methyltransferase (PIMT) is
suggested to play a role in the repair of aged protein spontaneously
incorporated with isoaspartyl residues. We generated PIMT-deficient
mice by targeted disruption of the PIMT gene to
elucidate the biological role of the gene in vivo.
PIMT-deficient mice died from progressive epileptic seizures with grand
mal and myoclonus between 4 and 12 weeks of age. An anticonvulsive
drug, dipropylacetic acid (DPA), improved their survival but failed to
cure the fatal outcome. L-Isoaspartatate, the putative
substrate for PIMT, was increased ninefold in the brains of
PIMT-deficient mice. The brains of PIMT-deficient mice started to
enlarge after 4 weeks of age when the apical dendrites of pyramidal
neurons in cerebral cortices showed aberrant arborizations with
disorganized microtubules. We conclude that methylation of modified
proteins with isoaspartyl residues is essential for the maintenance of
a mature CNS and that a deficiency in PIMT results in fatal progressive
epilepsy in mice.
Key words:
protein L-isoaspartyl methyltransferase; gene
targeting; isoaspartate; epilepsy; disorganized microtubules; aberrant
arborizations
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INTRODUCTION |
Asparaginyl and aspartyl residues
are the most labile amino acids subject to spontaneous degradation
reactions that modify aging proteins. This is attributable, in part, to
their susceptibility to the intramolecular attack of the side chain
carbonyl carbon by the adjacent backbone nitrogen, resulting in the
formation of a succinimide intermediate (Geiger and Clarke, 1987
). This intermediate ring can hydrolyze at either of its two carbonyl groups,
yielding both isoaspartyl (isomerization) and normal aspartyl residues
(McFadden and Clarke, 1987). The methyltransferase activity has been
widely detected in various organisms, which transfer the methyl residue
to the side chain carboxyl group of L-isoaspartyl and
D-aspartyl residues but not to normal
L-aspartyl residues (McFadden and Clarke, 1982; Aswad,
1984; Clarke, 1985; Ingrosso et al., 1989), and has been designated
protein L-isoaspartyl methyltransferase (PIMT) (Clarke,
1985). Experiments performed in vitro have demonstrated that
the incubation of synthetic L-isoaspartyl-containing
peptides with PIMT results in the conversion of at least 50% of the
peptide to the normal L-aspartyl-containing form (Johnson
et al., 1987; McFadden and Clarke, 1987). This finding led to the
hypothesis that the function of PIMT in vivo is to minimize
the accumulation of potential L-isoaspartyl residues in
long-lived proteins as proteins age. However, this hypothesis is only
based on data obtained by in vitro aging experiments,
whereas the in vivo function of PIMT is still unknown. To
clarify the biological role of PIMT, we disrupted the PIMT
gene by gene targeting and found that protein methylation is essential
for the maintenance of the CNS and that a deficiency of PIMT results in
fatal progressive epilepsy in mice.
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MATERIALS AND METHODS |
Construction of a targeting vector and production of
gene-disrupted mice. The C57BL/6 mouse genomic library in 
DASH
II was screened with human PIMT cDNA (Shirasawa et al.,
1995) as a probe. Out of 1 × 106 phages
screened, a recombinant phage containing genomic DNA of PIMT
locus was isolated. The targeting vector
pPNT-PIMT-neo was constructed by inserting a 0.79 kb PCR-amplified 3'-homologous fragment (short arm) into a
XhoI/NotI-restricted pPNT vector (Tybulewicz et al., 1991). The short arm was amplified from a genomic clone by PCR with a XhoI-anchored sense primer
(5'-ATCTCGAGTGCTGCACAAACAGGGCACATG-3') and a
NotI-anchored complementary primer
(5'-AAGCGGCCGCTCACATATCTTTCCACCTGC-3'). The 3'-NsiI site of
the 10.4 kb EcoRI-NsiI-restricted fragment was
converted to the BamHI site by ligating adapters
(5'-AGCTT-3' and 5'-GATCAAGCTTGGAT-3') and subcloned into the
EcoRI/BamHI-restricted targeting vector.
The ES cell line E14 (Hooper et al., 1987) was cultured, transfected,
and screened as described previously (Kitamura et al., 1991). After
transfection of the NotI-linearized
pPNT-PIMT-neor construct, 250 colonies resistant to both G418 and GANC were screened for the
homologous recombination event by PCR with a primer located downstream
of the short arm (5'-GGCACACAGAGACACCAAG-3') and a primer
complimentary to the PGK-neor gene
sequence (5'-ACTTGTGTACGCCAAGTGC-3') at an annealing
temperature of 65°C. The precise homologous recombination of
PCR-positive clones was confirmed by Southern blot analysis. Genomic
DNA from a PCR-positive clone was digested with BamHI and
XhoI and hybridized with a 1.2 kb SacI fragment
as a 5'-probe or digested with BamHI and EcoRI
and hybridized with a 0.73 kb BspHI/SspI fragment
as a 3'-probe (Fig.
1A). One ES clone
designated ES84 contained the expected PIMT-targeted allele.
Hybridization with the neor probe
verified the presence of a single copy of
neor gene for ES84. ES84 was then
injected into the blastocysts of C57BL/6 mice as described previously
(Bradly, 1987). Resulting male chimeras were mated to C57BL/6 females,
and their agouti offspring were screened by PCR and Southern blot
analysis to detect the heterozygous mutation in the genome using DNA
prepared from their tails. ES84 was able to transmit the
PIMT-targeted allele in the germline. The heterozygous mice
were intercrossed to produce homozygous offspring.
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Figure 1.
Targeted disruption of the PIMT gene.
A, top, Genomic organization of the
PIMT gene containing exons I-VII (closed
boxes). Middle, The targeted vector pPNT in
which the 10 kb EcoRI/NsiI and the 0.8 kb
HpaI/NsiI fragments of the
PIMT genomic clone were inserted into the
EcoRI/XbaI and
XhoI/NotI sites, respectively. Arrows indicate the direction of transcription. The
shaded boxes of tK and neo
genes represent the PGK-1 promoter. Bottom, The predicted structure of the PIMT locus after a targeting
event. Exon II, intron II, and exon III are replaced with the
neo gene. The fragments used for Southern blot and PCR
amplification are E, EcoRI;
B, BamHI; S,
SacI; Xb, XbaI;
Ns, NsiI; Hp,
HpaI; Xh, XhoI; and
N, NotI. B,
C, Southern blot analysis of genomic DNA extracted from
mouse tails. DNA was digested with BamHI and
XhoI and hybridized with the 5'-probe
(B). DNA was digested with BamHI and EcoRI and hybridized with the 3'-probe
(C). The sizes of wild-type (3.4 kb) and
disrupted (11.4 kb) alleles are shown (C). The
genotypes of mice are presented above the lanes.
D, RNase protection analysis of PIMT+/+,
PIMT+/ , and PIMT/ mouse brain and
testis. Ten micrograms of total RNA prepared from the indicated mouse
genotypes were subjected to RNase protection analysis probed by a
35S-labeled cRNA probe. Protected wild-type (365 bp) and
targeted (256 bp) transcripts were separated by a denaturing PAGE gel. E, Immunoblot analysis of PIMT+/+,
PIMT+/, and PIMT/ mouse brain and
testis. Upper, Lysates of homogenized tissues derived from the indicated mouse genotypes were subjected to SDS-PAGE and
blotted to a nitrocellulose membrane. The blot was probed with
anti-PIMT antibody. Lower, The bands (26 kDa)
corresponding to PIMT were measured by densitometry, and their
intensities were presented in a graph.
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RNase protection analysis and immunoblot analysis. Total RNA
was prepared from brains or testes from PIMT/ and
control mice by the guanidine-CsCl method as described (Chirgwin et
al., 1979). Ten micrograms of total RNA were hybridized with
32P-labeled cRNA antisense probe at 50°C overnight and
were digested with 40 µg/ml of RNase A and 2 µg/ml of RNase T1 at
37°C for 30 min using the PRAII kit (Ambion, Austin, TX). Protected
fragments were resolved on denaturing PAGE gels.
32P-labeled cRNA probe was prepared from the pGEM7Z vector
(Promega, Madison, WI) carrying the 365 bp mouse PIMT coding
sequence [nucleotides (nts) 203-567; D38023] by T7 RNA polymerase
according to the protocol of the manufacturer (Promega).
For Western blot analysis, brain tissues were homogenized with
Tris-saline (TS) buffer (50 mM Tris-HCl, pH 7.6, and 150 mM NaCl), treated with SDS sample buffer, and resolved on
15-22% gradient SDS-acrylamide gels. Proteins were electrophoresed to a polyvinylidene fluoride membrane (Millipore, Bedford, MA). The blot
was blocked in TS buffer with 3% gelatin, incubated with anti-PIMT
antiserum diluted 1:500 in TS buffer, washed briefly in TS buffer, and
incubated with biotinylated anti-rabbit secondary antibody (Vector
Laboratories, Burlingame, CA) diluted 1:500 in TS buffer, followed by
the avidin-biotin complex. Immunoreactions were visualized by color
development using 4-chloro-1-naphthol, and optical densities were
measured to determine the relative amount of PIMT in the
immunoblot.
Histopathological examinations, immunohistochemical analysis, and
electron microscopy. For routine morphological evaluations, tissues were dissected, weighed, and fixed in 4% paraformaldehyde in
PBS, pH 7.0. Paraffin sections were prepared by standard procedures and
stained with hematoxylin and eosin (H&E) or Nissl stain. For immunohistochemical staining of neurofilament (NF), the animals were
perfused with ice-cold acid and alcohol solution (95% ethanol and 5%
acetic acid), and extracted brain tissues were fixed in the same
solution. Eight micrometer paraffin sections were incubated with
anti-NF antibody (1:500 dilution) (Fukuda et al., 1997) at 4°C
overnight. After being washed with PBS, sections were incubated with
peroxidase-conjugated goat anti-rabbit IgG Fab fragment (1:100 dilution; Medical and Biological Laboratories, Nagoya, Japan). Immunoreactivity was developed in 50 mM Tris, pH 7.4, containing 0.01% DAB and 0.01% hydrogen peroxide. For
immunohistochemical staining of MAP-2, tissues were fixed in a 4%
paraformaldehyde and 30% sucrose mixture in PBS, pH 7.0, mounted in
OCT compound (Miles, Elkhart, IN) and were snap frozen in dry ice. Ten
micrometer cryostat sections were incubated with anti-MAP-2 antibody
(1:200 dilution; Amersham, Arlington Heights, IL). After washing with PBS, sections were incubated with FITC-conjugated anti-mouse
immunoglobulins (1:500 dilution; Amersham). Labeled sections were
sealed in mounting medium containing antifader (IMMUNON).
Immunoreactivity was visualized by confocal laser microscopy (MRC1000;
Bio-Rad, Hercules, CA). For electron microscopic analysis, mice were
perfused with a 2.5% glutaraldehyde and 1% paraformaldehyde mixture
in 0.1 M phosphate buffer, pH 7.0. Extracted tissues were
post-fixed with 0.1% OsO4 in 0.1 M phosphate
buffer for 1 hr, stained en bloc in 1% uranyl acetate, and embedded in
Epon 812 after dehydration with ethanol. Serial sections of 0.5 or 1 µm thickness from cerebral cortices from PIMT/ or
control mice were cut by a Reichert microtome, placed on a hormbar
membrane of single hole mesh (Synaptec), stained conventionally, and
observed with a JEOL 4000EX intermediate voltage electron microscope at
350 kV.
Methyltransferase activity and determination of isoaspartate and
D-amino acids. PIMT activity was measured essentially
as described (Lowenson and Clarke, 1991) with ovalbumin as a substrate. Tissues extracted from PIMT/ and control mice were
homogenized in 50 mM phosphate buffer, pH 7.0, containing 2 mM EDTA and 15 mM 
-mercaptoethanol, and the
protein concentration was determined by BCA assay. In assays, 100 µg
of ovalbumin was incubated in 30 µl of 0.2 M
sodium-citrate buffer, pH 6.0, with 10 µM
S-adenosyl-L-[14C]methionine
(2.15 GBq/mmol; CFA.360; Amersham) and in 20 µg of tissue homogenate
at 37°C for 30 min. Each reaction was quenched with 50 µl of 0.2 M NaOH in 1% SDS, and immediately a 60 µl aliquot was
spotted on a piece of filter paper. The paper was wedged into the neck
of a 10 ml scintillation vial containing ACSII (Amersham), which was
then capped and allowed to equilibrate at room temperature for 3 hr.
Specific activity was presented as picomoles of 14C per
minute per milligram of protein. Data were presented as an average ± SD of six experiments.
Isoaspartic acid residues were quantified using the ISOQUANT protein
deamidation kit (Promega) with isoaspartyl-DSIP as a standard substrate
as described (Paranandi et al., 1994). Brain homogenates were prepared
as described above. Isoaspartic acid residues was presented as
pmol/µg of homogenate. Data were presented as an average ± SD
of triplicate experiments.
D-Amino acids were quantified essentially as described
(Hayashi and Sasagawa, 1993). Brain tissues from PIMT/
and control mice were hydrolyzed by adding 300 µl of 6N HCl and
incubating at 110°C for 6 hr. Samples were then dried, dissolved in
500 µl of DDW, and derivatized with
(+)-1-(9-fluorenyl)-ethylcholoroformate (Fluka, Neu-Ulm, Germany).
Derivatized samples were analyzed by HPLC (Hitachi, 655A12). The
amounts of D-amino acids were presented as the ratio of the
amount of D-amino acid to the combined amount of
D- and L-amino acid.
Video monitor of epileptic attack. Behavior and epileptic
attacks of PIMT/ mice were monitored and recorded for 24 hr by home video apparatus. The type and frequency of epileptic
seizures were visually diagnosed using the recorded video.
Dipropylacetic acid (DPA) sodium was orally administered by dissolving
3 mg of DPA in 3 ml of drinking water.
Electroencephalograph. When animals were under anesthesia
with Ketamin (20 mg/ml) and Xylazine (9 mg/ml), four epidural silver ball electrodes, 0.1 mm in diameter, were implanted in the left frontal, right frontal, left occipital, and right occipital epidural space via holes in the skull by the implantation method described previously (Nakano et al., 1993, 1994). Five hundred microliters of 5%
glucose in physiological saline were intraperitoneally administered to
aid rapid postoperative recovery from anesthesia, and all examined mice
survived the operation. A stainless electrode was placed subcutaneously
near the nose as a reference for monopolar recordings. A cassette
connector was placed on the back with the outlet leads connected to the
preamplifier of recording apparatus as described previously (Nakano et
al., 1994). Electroencephalographs (EEGs) including four monopolar
recordings and four bipolar recordings were recorded everyday for 2 hr
between 10 A.M. and 5 P.M. from the second day after the operation. All
epileptic spikes successfully recorded were spontaneous cortical
discharges without any evocation.
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RESULTS |
Generation of PIMT knock-out mice
A 15 kb PIMT genomic clone obtained from a mouse
C57BL/6 genomic library was used to construct a targeting vector
(pPNT-PIMT) for homologous recombination by
positive-negative selection (Mansour et al., 1988), as shown in Figure
1A. Exons II and III of the PIMT gene (nt
75-311; GenBank/EMBL accession number D38023) were replaced by the
PGK-neor cassette in inverse orientation,
which disrupted the gene by deleting a segment encompassing from
Lys19 to Met64 of the
PIMT transcript. The disrupted gene failed to be
productively spliced from exons I to IV in frame (Romanik et al.,
1992). NotI-linearized pPNT-PIMT was
electroporated into E14 ES cells, and neomycin-resistant clones were
selected using G418. After selection, 250 ES clones were isolated, of
which one clone carried the expected targeted recombination event as
determined using the 3'-probe outside of the targeted construct (Fig.
1A).
Targeted ES cells were injected into C57BL/6 blastocysts, and three
chimeric animals were generated. One male transmitted the targeted
allele to germline cells. Heterozygous F1 animals were interbred to
generate homozygous PIMT-deficient mice. The genotype was determined
using PCR (Fig. 1A) and confirmed by Southern blot
analysis (Fig. 1B,C). The 5'-probe detected a 7.4 kb
BamHI fragment of the wild-type allele and a 8.9 kb
BamHI/XhoI fragment of the knock-out allele in
the BamHI/XhoI double-digested genome (Fig.
1B). The 3'-probe detected a 3.4 kb
BamHI/EcoRI fragment in a wild-type allele and a
11.4 kb in a knock-out allele (Fig. 1C). These results are
consistent with the predicted replacement by the
PGK-neor cassette (Fig.
1A). In addition, RNase protection analysis of brain
and testis RNA probing the 365 bp fragment (nt 203-567) of mouse
PIMT (exons II-VI) showed the 365 bp fully protected transcript in RNAs prepared from brains and testes of
PIMT+/+ and PIMT+/ mice, whereas the 256 bp
partially protected transcript (nt 312-567) was found in
PIMT+/ and PIMT/ mice (Fig.
1D). Finally, immunoblot analysis of brain and testis
lysates using anti-PIMT antibody (Shirasawa et al., 1995) showed the
strong 26 kDa band in PIMT+/+ mice and a less intense band
in PIMT+/ mice, but no corresponding band was detected in
the lysates from PIMT/ mice (Fig. 1E,
upper panel). The intensity of immunoreactivity in PIMT+/ mice was approximately half of the intensity
detected in PIMT+/+ mice (Fig. 1E,
lower panel). The generation of
PIMT/ animals followed Mendelian segregation, indicating
that the homozygous genotype was not embryonically lethal.
The L-isoaspartate but not the D-aspartate
residue is increased in the brain of PIMT-deficient mice
To confirm the deficiency of enzymatic activity in
PIMT/ mice, we assayed the lysates prepared from brain,
kidney, testis, and eye of PIMT+/+, PIMT+/, and
PIMT/ mice for methyltransferase activity by assessing
the incorporation with [14C]methyl residues of
S-adenosyl-L-methionine (SAM) into ovalbumin (Fig. 2A). The lysate
prepared from PIMT+/+ mice showed a specific activity of
14.6 ± 2.5 (pmol/min/mg of protein, average ± SD; n = 3) in testis, 12.2 ± 0.99 in the brain
(n = 3), 3.60 ± 0.58 in the kidney
(n = 3), and 1.46 ± 0.16 in the eye
(n = 3; Fig. 2A). The lysate from
PIMT/ mice showed no enzymatic activities in the testis,
brain, and eye but reduced activity in the kidney. The results
suggested that major enzymatic activities for methyltransferase were
completely deficient in examined tissues from PIMT/
mice, except in the kidney where other minor methyltransferase activity may exist. The results also showed that PIMT+/ mice
exhibited approximately half of enzymatic activity of
PIMT+/+ mice (Fig. 2A), which was
consistent with the expression of PIMT in immunoblot analysis (Fig.
1E).
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Figure 2.
PIMT specific activity was deficient in
PIMT/ mice, and the amount of L-isoaspartyl
residues but not that of D-aspartyl residues was increased
in the brains of PIMT/ mice. A, Lysates prepared from brains, kidneys, testes, and eyes of the indicated mouse
genotypes were assayed for PIMT-specific activity. Each bar represents the mean ± SD for three 5-week-old
mice. B, L-Isoaspartyl residues were
measured in the lysates prepared from brains of the indicated genotypes
by the incorporation of [3H]methyl residues by
recombinant PIMT. Each bar represents the mean ± SD for three 5-week-old mice. The significance of differences (*p < 0.001) was determined by a Student's
t test. C, The ratio of
D-amino acids to L-amino acids was measured in
the brain (gray matter) of the indicated genotypes. No significant
differences were found between PIMT+/ and
PIMT/ mice in the amount of D-amino acid
divided by the total amount of D-amino acid plus
L-amino acid (D/D + L ratios) for total D-amino
acids, D-aspartate, D-methionine, D-tyrosine, D-phenylalanine,
D-histidine, and D-arginine.
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We next investigated whether the substrate molecules were accumulated
in PIMT-deficient mice. In previous studies in vitro, PIMT
reacted in a stoichiometric manner to L-isoaspartate
residues in a synthetic peptide but in a substoichiometric manner to
D-aspartate residues (O'Connor et al., 1984). However,
still undefined is whether D-aspartate and
L-isoaspartate residues are bona fide substrates for PIMT
in vivo. Therefore, we investigated the accumulation of
substrates with L-isoaspartate and D-aspartate
residues in the brain tissues from PIMT/ mice. The brain
lysates prepared from PIMT+/+, PIMT+/, or
PIMT/ mice were assayed for the capacity of methyl
incorporation by recombinant PIMT (Fig. 2B). The
results showed that the amount of isoaspartyl residues was increased
ninefold in PIMT/ mice (0.703 ± 0.145 pmol/mg of
lysate, average ± SD; n = 3) compared with
PIMT+/+ mice (0.075 ± 0.018; n = 3) or
PIMT+/ mice (0.079 ± 0.002; n = 3).
On the other hand, the ratio of D-aspartate to
D-aspartate plus L-aspartate was not increased
in the brain from PIMT/ mice compared with
PIMT+/ mice, as well as the ratios of other
D-amino acids including D-methionine,
D-tyrosine, D-phenylalanine, D-histidine, and D-arginine (Fig.
2C). These results suggested that the proteins carrying
L-isoaspartate but not D-aspartate residues are
the in vivo substrates for PIMT.
Clinicopathological phenotype of PIMT/ mice
PIMT/ mice were born at the normal Mendelian
frequency from heterozygote crosses in 129 × the C57BL/6
background. PIMT/ neonates developed normally and were
viable, but their weights were ~35% smaller than PIMT+/
or PIMT+/+ mice until 7 weeks after birth (data not shown).
However, PIMT/ mice all died between 4 and 12 weeks of
age (Fig. 3A, solid
line with closed circles). A macroscopic
study on postmortem examinations has revealed that the brains of
PIMT/ mice were 15% heavier than were those of PIMT+/ mice (509.6 ± 37.9 vs 459.2 ± 28.5 mg;
p < 0.0001; Figs. 3B,
4B), and the spleens of
PIMT/ mice were 28% smaller than were those of
PIMT+/ mice (66.5 ± 16.8 vs 91.3 ± 38.8 mg;
p < 0.05). Nevertheless, a routine histological
examination failed to reveal any apparent pathological abnormality
causing death, excluding the possibility that PIMT/ mice
had died from a chronic, progressive pathological process of the heart,
lung, or other structures examined. Because gross appearances of some
PIMT/ mice (~20%) showed a kyphosis of the spine
(Fig. 4A), we checked their bones by x ray.
Radiological studies, however, failed to show any abnormality in the
bone of the vertebrae, skulls, and limbs, suggesting that the kyphosis
of the spine may not result from a bone abnormality but rather from the
weakness of the muscle providing a support for the vertebral columns,
as seen in muscular dystrophies (Wilkins and Gibson, 1976).
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Figure 3.
Survival curve and brain weight of
PIMT/ mice. A, Kaplan-Meier survival
curves of PIMT/ (closed circles) and
PIMT+/ (open circles) mice. The
survival of PIMT/ mice was improved by antiepileptic medication with DPA (closed diamonds). B,
Brain weights of 24 PIMT/ mice (closed
circles) and of 24 littermate control mice (PIMT+/, open circles) plotted versus
their age. An upper regression line represents the
weights of brains from PIMT/ mice with a slope of
17.959, and a lower regression line represents those of
PIMT+/ mice with a slope of 4.375.
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Figure 4.
Gross appearance and brain pathology of
PIMT/ mice. A, Picture of 5-week-old mice
derived from intercross-breeding of PIMT heterozygous mice.
PIMT/ (left) and
PIMT+/ (right). B, Gross appearance of brains of 5-week-old PIMT/
(left) and PIMT+/+ (right)
mice. C, D, Nissl staining of neurons in
layer 5 of the precentral cortex from PIMT/
(C) and PIMT+/+
(D) mice. Magnification: C,
D, 400×.
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A routine microscopic examination of the brain sections from
PIMT/ mice using hematoxylin-eosin staining showed the
proportional enlargement in all examined areas including gray matter
and white matter of the cerebral cortex, hippocampus, striatum,
thalamus, and cerebellum. We then investigated the number of neurons or astrocytes in the dentate gyrus of the hippocampus or cerebral cortices
(Fig. 4C,D, PIMT/ and PIMT+/+,
respectively) and the number of oligodendrocytes in given microscopic
fields of the white matter, revealing no significant increase in the
number of neurons or astrocytes (Fig. 4C,D) or of
oligodendrocytes in PIMT/ mice (data not shown); these
observations imply that enlargement of the brain was not because of the
increased number of neurons as described in CPP-32-deficient mice
(Kuida et al., 1996) or because of the gliosis often observed after
brain injuries or inflammations but may be because of the increased
volume of extracellular matrix or the increased extrasomatal structures
such as neural fibers in the brain of PIMT/ mice. In
Nissl sections, cortical pyramidal neurons of bizarre morphology with
an abnormally ballooned nucleus and clearly demarcated nucleolus were
found in layer V of PIMT/ mice (Fig. 4C) but
not in PIMT+/+ mice (Fig. 4D). However, the appearance of these morphologically abnormal neurons in the precentral cortex of PIMT/ mice suggested that this
abnormality would not be attributable to the generalized enlargement of
the brain but may rather be related to myoclonic seizures observed in
this mice (see below).
We then investigated blood chemistry, lipid metabolism, electrolyte
balance, and hormones in pooled sera from PIMT/ as well as PIMT+/+ mice. In blood chemistry, PIMT/
mice showed normal levels for total protein, albumin,
glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase,
alkaline phosphatase, and cholinesterase but decreased levels for
lactate dehydrogenase (996 vs 1306 IU/l in PIMT/ vs
PIMT+/+), creatine phosphokinase (1138 vs 2135 IU/l), and
ferritin (2.5 vs 5.2 ng/ml). In lipid metabolism, PIMT/ mice showed normal levels for phospholipid and free fatty acid but an
increased level for triglyceride (70 vs 52 mg/dl) and decreased levels
for total cholesterol (86 vs 116 mg/dl) and esterified cholesterol (75 vs 105 mg/dl). In electrolytes, PIMT/ mice showed normal
levels for Na, K, Cl, Ca, and creatinin but showed a decreased phosphate level (9.8 vs 12.1 mg/dl) and an increased blood urea nitrogen level (30.3 vs 19.7 mg/dl). In hormones examined so far, PIMT/ mice showed normal levels for free T3 and T4 but
increased levels for aldosterone (830 vs 430 pg/ml) and corticosterone
(0.24 vs 0.12 mg/ml). All these data may suggest a catabolic state of PIMT/ mice, whereas a decrease in CPK may suggest a
decreased amount of body muscle.
PIMT-deficient mice die from generalized
tonic-clonic seizures
PIMT-deficient mice showed a shortened survival (Fig.
3A). Approximately half of PIMT/ mice
spontaneously died from 4 to 5 weeks of age, and the remaining half
died at a rather constant rate until 13 weeks of age (Fig.
3A, left solid line with closed circles). The fact that few PIMT/ mice die
before 4 weeks of age in spite of abnormalities such as small body size
suggests that the abnormality causing a sudden death may be related to maturation defects or a failure in the maintenance of matured organ
systems rather than to developmental defects. We then monitored the
clinical features of PIMT/ mice for 24 hr by video
camera to catch the clinical feature at the last moment of death. To our surprise, PIMT/ mice died from grand mal-type
epileptic attacks with jumping fits or refractory status epilepticus.
In their video recording, PIMT/ mice showed various
types of epileptic seizures including jumping fits, forelimb clonus,
running fit, tonic-clonic (GTC) seizures, and myoclonic jerks of the
limbs and the trunk. These clinical features, taken together with the following electroencephalographic data, implied that the underlying basis for the epilepsy may be attributable to an increased neuronal excitability in cerebral cortices, hippocampi, the limbic system, and
the brainstem.
We next recorded EEGs of PIMT/ mice to determine the
type of epilepsy. EEGs from three PIMT/ mice were
successfully recorded and compared with that of control mice. Three
PIMT/ mice manifested clinical convulsions with EEG
recordings of paroxysmal cortical discharges with spikes and waves as
shown in Figure 5, whereas control
PIMT+/+ mice had no clinical convulsions or abnormal
cortical discharges. A typical EEG recording of grand mal seizure was
presented in Figure 5 in which PIMT/ mice clinically
showed static tonic phase (Fig. 5, phase A), dynamic
tonic phase (phase B), long clonic phase
(phase C), and a jumping fit
(phase D) followed by the posticteric cortical
suppression with long duration. Static tonic seizure started abruptly
in all bipolar and monopolar leads as presented in Figure 5. After the
generalized spikes in tonic phase, typical spikes and waves were
recorded in the left frontal lead (Fig. 5, phase C)
for >20 sec when the mouse showed a forelimb clonus in the standing
position on hindlimbs. Although these spikes and waves failed to spread
to the other parts of the brain during clonic phase, the spikes became
generalized again in phase D when the mouse showed an abrupt jumping
fit (Fig. 5, phase D), followed by a deep coma of long
duration. The epileptic animal presented characteristic clinical
features in its long-lasting posticteric period when respiratory
arrests were often observed in PIMT-deficient mice. As shown in Figure
5, only muscle activity corresponding to respiratory movement was
recorded in all EEG leads, suggesting that electrical activity in the
brains of PIMT/ mice was severely suppressed in this
period. The fact that PIMT/ mice showed the respiratory
arrests after grand mal attacks on the recorded video monitor was well
explained by this EEG record. We suggest based on these data that the
respiratory suppression after the epileptic seizure may be the direct
cause of death in PIMT/ mice, although alternatively
fatal accidents such as arrhythmia could arise in this period.
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Figure 5.
EEG recordings of PIMT/ mice. A GTC
seizure of a PIMT/ mouse. The beginning of GTC seizure
is indicated as Onset of GTC. During the initial static
tonic phase (A), the mouse stopped moving and
stayed in a prone posture with strong lordosis of the spine and
progressive elevation of the tail. During the following dynamic tonic
phase (B), the mouse squeaked once and fell over
on each side with dynamic torsion of the trunk and tonic elevation of the tail, followed by an opistotonic posture. During the clonic phase
(C), the mouse resumed a prone posture,
immediately stood on the hindlimbs, and continued the periodic or
clonic movements of the forelimbs. The EEG showed a continuous
spike-and-wave complex on left frontal recordings, whereas the other
monopolar recordings showed continuous rapid rhythm. During a jumping
fit (D), the mouse began to walk and jumped
several times. When the GTC seizure terminated, the mouse become
immobilized, accompanied by a deep respiration. During this phase, the
baseline fluctuation in monopolar recordings reflected respiratory
movements (up arrowhead), whereas the bipolar recordings
showed strong postictal cortical suppression. L, Left; R, right; F,
frontal; O, occipital.
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We next explored the possibility that DPA sodium (valproate), a widely
used antiepileptic drug, could prevent the seizure of
PIMT/ mice and thus elongate the survival of
PIMT/ mice. A therapeutic dose of DPA (3 mg/ml in
drinking water) was administered orally to PIMT/ as well
as to PIMT+/+ mice, and the frequency of epileptic seizures
was checked on the video monitor. Serum concentrations of DPA checked
in several treated mice were 20-50 µg/ml. The recordings on the
video monitor showed that the frequency of epileptic seizures diagnosed
on the video monitor was significantly reduced (minimally seen) in
PIMT/ mice until 7-8 weeks, and the survival of
PIMT/ mice was significantly elongated by an average of
4 weeks (Fig. 3A, solid line with
closed squares). However, after 8 weeks of age,
PIMT/ mice showed the reluctant epileptic attacks in
spite of the therapeutic dose of DPA and finally died either from
status epileptics or the toxic suppression of DPA before 12 weeks of
age (Fig. 3A). Based on the clinical effects of DPA in the
survival of PIMT/ mice, we reconfirmed that the direct
cause of death in PIMT-deficient mice was attributable to epileptic
attacks that were initially controlled by DPA but became uncontrollable
by 12 weeks of age.
Abnormal dendritic arborizations in pyramidal neurons of
PIMT-deficient mice
To define the pathological basis for the abnormal neuronal
excitability, we examined the brain morphology of PIMT/
mice. As noted earlier, there was a 15% increase in brain size in
PIMT/ mice compared with wild-type littermates (Fig.
4B) without significant macroscopic abnormalities
such as hydrocephalus, brain edema, or brain tumor. We next
investigated the brain size in various stages of PIMT/
mice and compared the sizes with those of PIMT+/ mice
(Fig. 3B). Surprisingly, the brains of PIMT-deficient mice began to increase in volume after 4 weeks of age when the basic structure of the brain was, in general, supposed to have been formed.
The brain size of PIMT/ mice increased continuously with
age until it finally showed approximately a 25% increase on average at
10 weeks of age (Fig. 3B). The abnormal increase in the
brain size seems to coincide with the occurrence of epileptic attacks
as well as the sudden death of PIMT/ mice (Fig.
3A,B), suggesting that the morphological analysis in the
brain may shed light on the pathological basis for the epileptogenesis
of PIMT-deficient mice.
To examine further the neuronal structure of the brain of
PIMT/ mice, we investigated the morphology of dendrites,
somata, and synapses of the neurons by localizing the
immunoreactivities for neurofilament (NF), MAP-2, and synaptotagmin. As
shown in Figure 6, the immunostaining for
NF in cerebral cortices of PIMT/ mice showed the
deformed apical dendrites projecting to the surface of the cerebrum
(Fig. 6B) as compared with the straight projections of the control mice (Fig. 6A). The pyramidal neurons
with abnormal dendrites were chiefly localized in lateral region of the
parietal cortex and began to appear at 5 weeks of age with varying
degrees of deformities. In high-power fields, the immunoreactivity for NF showed a twisted conformation in apical dendritic processes of
pyramidal neurons in layers II and III of the cerebral cortices from
PIMT/ mice, whereas the horizontal dendritic fibers in the cerebral cortices showed less-twisted structures (Fig.
6D). Immunolocalization of MAP-2 in pyramidal neurons
by confocal microscopy confirmed further that the apical dendrites of
the pyramidal cells in PIMT/ mice aberrantly wound and
sometimes bifurcated in the proximal portions (Fig.
6F), a process not observed in the control mice (Fig.
6E). The enigma that still remained to be answered is
whether these morphologically abnormal dendrites are functional or
dysfunctional and whether the increase in functional dendrites confers
the pathological basis for epilepsy. To address these issues, we
examined the immunolocalization of synaptotagmin and assessed whether
these dendrites retain functional synapses. The result showed that the
immunoreactivity for synaptotagmin in PIMT/ mice was not
increased or decreased, with a similar distribution in the brain of
PIMT/ mice compared with that of the control mice (data
not shown), implying that the epileptogenesis of PIMT/ mice was not a simple case of increased functional dendrites but an
aberrant dendritic arborization that may, in large part, be nonfunctional because of the cytoskeletal abnormalities (see below). In
this context, it is noteworthy to add that aberrantly arborized mossy
fibers with the synaptic reorganization were found in epileptic human
temporal lobes (Sutula et al., 1989).
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Figure 6.
Aberrant dendritic arborizations in cortical
neurons from PIMT/ mice. Localization of neurofilament
(NF) immunoreactivity in cerebral cortex from PIMT+/+
(A, C) and PIMT/
(B, D) mice is shown. Apical dendrites of
pyramidal neurons in layers II and III of the cerebral cortex from
PIMT/ mice showed the twisted configurations
(B, D). Localization of MAP-2
immunoreactivity in cortical pyramidal neurons from
PIMT+/+ (E) and
PIMT/ (F) mice are
shown. In PIMT/ mice, apical dendrites aberrantly
wind and bifurcate in the proximal portion
(F) Magnification: A,
B, 100×; C, D, 500×;
E, F, 1600×.
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To analyze the dendritic abnormality further, we investigated the
ultrastructure of dendrites, axons, and somatic structures of pyramidal
cells by electron microscopy. As shown in Figure 7, the diameter of axons in
PIMT/ mice was slightly decreased (Fig. 7B)
as compared with that in the control mice (Fig. 7A). In an
axon hillock of a pyramidal cell, Golgi apparatus
(g) and smooth (s) and rough
(r) endoplasmic reticulum were aberrantly arranged in the
somata, so that the distended hillock was compressed to the nucleus in
PIMT/ mice (Fig. 7D) as compared with the hillock of PIMT+/+ mice (Fig. 7C). In a higher
magnification of dendrites, the density of microtubules was locally
increased to form high density bundles in the dendritic sections of
PIMT/ mice (Fig. 7E,F, arrows),
whereas the structure of microtubules was evenly distributed in those
of PIMT+/+ mice. In PIMT/ mice, microtubular
bundles were unevenly or peripherally localized inside the dendritic
fibers (Fig. 7E), which was closely associated with abnormal
winding of apical dendrites. The result suggests that the organization
of microtubules was altered in PIMT/ mice and that
structural alterations in dendritic and axonal cytoskeletons may give
rise to abnormal dendritic arborization of pyramidal neurons in
PIMT/ mice.
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Figure 7.
Electron microscopy of cortical neurons from
PIMT+/+ and PIMT/ mice. A,
B, Pyramidal cells in layer III of the cerebral cortex
from PIMT+/+ (A) and
PIMT/ (B) mice. Both axonal
(a) and dendritic (d)
processes were observed. Abnormally arborized dendrites surrounding the
somata in PIMT/ mice are indicated by
arrows in B. The large
boxes indicate areas shown in subsequent views.
C, D, Higher magnification view of an
axon hillock of the pyramidal cell from PIMT+/+
(C) or PIMT/
(D) mice. The rough (r) or
smooth (s) endoplasmic reticulum and developed
Golgi apparatus (g) were observed.
E, F, Higher magnification view of a
dendrite from a PIMT/ mouse. High density bundles of
microtubules (arrows) were observed in the peripheral
dendritic section of a pyramidal neuron from a PIMT/
mouse. Magnification, A, B, 2100×;
C, D, 7500×; E, 14,000×;
F, 35,000×.
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DISCUSSION |
In this paper, we demonstrated that protein methylation is
essential for the maintenance of neuronal activity in the CNS and that
deficiency of protein methylation leads to fatal progressive epileptic
disease. In spite of the fact that PIMT is phylogenetically conserved
in various organisms including vertebrates, bacteria, fungi, and plants
(Johnson et al., 1991), its biological significance has not been so far
well understood. In fact, the repair of damaged aged proteins has been
suggested as a biological function of PIMT, this implication solely
based on in vitro experiments with various substrates
including calmodulin, synapsin, myelin, dentine, and crystalline
(Johnson and Aswad, 1985; Potter et al., 1993; Paranandi and Aswad,
1995). The fact that PIMT is highly expressed in various stages of
tissues including embryonic and neonatal brains (Shirasawa et al.,
1995) suggests role(s) of PIMT in the brain and other tissues in
addition to the repair of aged proteins. In this context, the epileptic
phenotype of knock-out mice was unexpected but suggestive for the
forthcoming studies on PIMT. We discuss here the biochemical alteration
of aspartyl residues and the epileptogenic mechanism(s) of
PIMT-deficient mice.
L-Isoaspartyl but not D-aspartyl residues
accumulate in the brain of PIMT-deficient mice
Previous studies on the specificity of PIMT using synthetic
peptides showed that PIMT methylates L-isoaspartyl residues
(KASA-L-isoD-LAKY) at a Km value of
0.52 ± 0.08 µM and D-aspartyl
residues (KASA-D-D-LAKY) at a Km
value of 2700 ± 400 µM with the stoichiometric
ratio of D-aspartate
Km/l-isoaspartate
Km/l being 5190-fold (Lowenson and Clarke, 1992). Based on the computer simulation with these values, Lowenson predicted that an average 100 kDa protein carrying 35 L-asparagine and 52 L-aspartate residues,
without the methyltransferase activity, should generate 0.52 residues
of L-isoaspartate, 0.008 residues of
D-aspartate, and 0.029 residues of
D-isoaspartate per molecule in 10 d. The addition of
PIMT activity to this mathematical simulation led to the reduction of
L-isoaspartate to <0.0036 but the increase of
D-aspartate up to 0.020 in 10 d (Lowenson and Clarke,
1992). In the present study, we showed that PIMT represents the major
protein methyltransferase in vivo in the brain, testis, and
eye, which can transfer the methyl residues into the synthetic peptides
carrying L-isoaspartate or ovalbumin (Fig. 3A).
Furthermore, we demonstrated that L-isoaspartyl residues
were significantly accumulated in the brain from PIMT/
mice (Fig. 3B) as well as in the testis, heart, muscle,
spleen, and eyes of PIMT/ mice (data not shown) but not
in those tissues from PIMT+/ or PIMT+/+ mice.
On the other hand, the D-aspartate residue was not
increased but rather decreased in the brain from PIMT/
mice in comparison with those of PIMT+/ mice (Fig.
2C). These in vivo data are fully compatible with
the previous hypothetical simulation that the complete deficiency of
PIMT activity theoretically leads to the accumulation of
L-isoaspartyl residues but a slight decrease in D-aspartyl residues in damaged proteins (Lowenson and
Clarke, 1992).
To characterize the substrate proteins accumulated in the brain from
PIMT/ mice, we labeled brain lysate prepared from
PIMT/ mice with [14C]methyl-SAM by
recombinant PIMT and resolved it on acidic PAGE gel. The labeled
molecules in brain lysate of PIMT/ mice were fractionated in a smear pattern with varying intensities detected; the
pattern of these intensities was, in large part, similar to that of
control mice, but the intensities in PIMT/ mice were significantly stronger than those of the control (data not shown), suggesting that the substrates for PIMT may be widely distributed in
various proteins, in which the equilibrium among
L-aspartate, L-isoaspartate,
D-aspartate, and D-isoaspartate in modified
aspartate or asparagine residues was drastically shifted to
L-isoaspartate in the substrate molecules of
PIMT/ mice. Further biochemical investigation on
PIMT-deficient mice would clarify the biological significance of the
shifted equilibrium in modified aspartate residues of the substrate
molecules in vivo.
PIMT deficiency results in a progressive epilepsy with grand mal
and myoclonus
Because 10-15% of human epilepsies are suggested to have
genetic backgrounds, the identification of mutations in causal genes can be used to define epileptogenic mechanisms. Several human epilepsy
genes have been mapped, and some of them were identified recently
(Silvestri et al., 1992; Shiang et al., 1993; Steinlein et al., 1995;
Pennacchio et al., 1996). However, the causal genes for the majority of
epilepsies still remain to be identified (Phillips et al., 1995; Ryan,
1995; Serratosa et al., 1995; Zara et al., 1995; Virtaneva et al.,
1996). Moreover, these efforts are complicated not only by clinical and
genetic heterogeneity but likely by polygenic inheritance as well. In
mice, a number of single-gene mutations that cause epilepsy have been
identified by the study of various knock-out mice (Brusa et al., 1995;
Li et al., 1995; Toth et al., 1995; Waymire et al., 1995; Matsumoto et
al., 1996; Rothstein et al., 1996; Brennan et al., 1997; Homanics et
al., 1997; Kim et al., 1997; Luthi et al., 1997), and it is hoped that
the identification and molecular analysis of these genes will provide
insights into human epilepsy. We show in the present studya mouse model
for fatal progressive epilepsy with grand mal and myoclonus and discuss possible novel epileptogenic mechanism(s).
In this study, several lines of evidence strongly suggested that
epilepsy was a direct cause of death in PIMT-deficient mice. First, the
anticonvulsive drug DPA decreased the frequency of seizures and
prolonged the survival of PIMT/ mice. Second, the video
monitor indicated that PIMT/ mice showed grand mal
attacks and myoclonic seizures and died soon after fatal convulsion
attacks or from refractory status epilepticus. Third, the postmortem
pathology showed otherwise normal tissues in hematoxylin-eosin
staining. EEG study indicated that PIMT/ mice showed
generalized tonic-clonic seizure with typical spikes and waves. Taken
together, PIMT-deficient mice can be an animal model for a progressive
myoclonus epilepsy (PME). PME is a clinical entity with myoclonic
seizures, tonic-clonic seizures, and progressive neurological
dysfunction such as ataxia or dementia and is classified into two major
forms, Unverricht-Lundborg disease and Lafora's disease. Recently,
point mutations in the cystatin B gene have been proven to be
responsible for Unverricht-Lundborg disease (Pennacchio et al., 1996).
The causal gene for Lafora's disease, on the other hand, has been
mapped to human chromosome 6q23-25 (Serratosa et al., 1995) but has
not yet been identified. Interestingly, human PIMT gene was
mapped to chromosome 6q22.3-6q24 (MacLaren et al., 1992). Furthermore,
the clinical profile of PIMT-deficient mice is consistent with that of
Lafora's disease that develops neurological symptoms during late
childhood and leads to a fatal outcome within a decade of the first
symptom (Elliott et al., 1992). It is reasonable to speculate that a
mutation in the PIMT gene may cause progressive myoclonus
epilepsy of the Lafora type in affected pedigrees. Further genetic
study on such pedigrees would clarify the causative relationship
between PIMT gene mutation and Lafora's disease.
Cytoskeletal abnormality and accumulation of modified proteins with
isoaspartyl residues
To date a number of knock-out or transgenic mice showing epileptic
phenotype have shed light on the molecular basis for epileptogenesis (Noebels, 1996). These include (1) ion channel genes such as mkv1.1 or
Weaver, (2) genes regulating the synaptic release of
neurotransmitters such as synapsin I and II or CAMKII, (3) synaptic
receptor genes such as GluRB or 5-HT2C gene, and (4) genes
associated with axonogenesis or synaptogenesis such as GAP-43. In our
experimental model, the biochemical alteration such as the accumulation
of isoaspartyl residues in the brain was clearly associated with the
onset of seizure in PIMT/ mice. It is then speculated
that some molecule(s) modified with isoaspartyl residues may confer the
epileptogenic basis in PIMT/ mice. However, it is
practically difficult to identify the responsible epileptogenic
molecule(s) by means of a biochemical approach because a number of
molecules were radiolabeled by recombinant PIMT in the brain lysate
prepared from PIMT/ mice.
Pathological analysis strongly suggested that the enlargement of the
brain observed in PIMT-deficient mice after 4 weeks of age may be
related to the progression of fatal epilepsy (Fig. 3). However,
morphological analyses of light (Fig. 4B) and
electron (Fig. 7) microscopic examinations as well as
immunohistochemical staining for NF, MAP-2, and synaptotagmin studies
(Fig. 6) revealed the swollen pyramidal neurons in layer V of the
precentral cortex (Fig. 4C) and the abnormal arborizations
of apical dendrites of cortical pyramidal neurons in the parietal lobe
of PIMT/ mice (Fig. 6). This abnormality could, in part,
contribute to the enlargement of the brain but fail to explain the 15%
increase in the brain size of PIMT/ mice, suggesting
that morphologically invisible alterations such as a generalized
increase of extracellular matrix may contribute to the enlargement of
the brain in PIMT-deficient mice. Further neurochemical investigation
on basic components such as MBP, tubulin, neurofilament, and actin or
sphingolipids such as sphingomyelins and cerebrosides may shed light on
the biochemical basis for the enlargement of the brain.
EM studies showed the abnormal microtubular organization in the
dendrites of pyramidal neurons. The abnormality is especially intriguing because PIMT immunolocalizes to neurofibrillary tangles (NFTs) in the brain of Alzheimer's disease (T. Shirasawa, unpublished observations). Because NFT is chiefly composed of abnormally
phosphorylated tau, it is suggested that tau is one of the candidate
substrates for PIMT in degenerating neurons. We then purified tau from
PIMT/ mice and investigated the isomerization of tau.
L-Isoaspartyl residues in tau purified from
PIMT/ mice were increased 2.7-fold compared with that
from PIMT+/+ mice (8.60 vs 3.23 pmol/µg protein), indicating that tau is an in vivo substrate for PIMT.
Because microtubule-associated proteins (MAPs) such as tau modulate and regulate the formation of microtubules in neuronal processes, one
possible mechanism for the disorganization of microtubules may be the
dysfunction of MAPs because of the protein isomerization. In addition,
Najbauer et al. (1996) recently reported that tubulin was also
isomerized in vivo as well as in vitro,
suggesting as another possibility that microtubules fail to organize
properly in the neural processes because of the isomerization of
tubulin itself. The assembly and disassembly of microtubules in
PIMT/ fibroblasts is now being investigated in our
laboratory using a GFP-Tau34 construct (Ludin et al., 1996) to address
these issues. As microtubules are involved in the transport of various
signal-transducing molecules such as neurotrophines or synaptic
molecules, the dysfunction of the transport system may confer the
alteration of neuronal excitability in PIMT-deficient mice.
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FOOTNOTES |
Received July 28, 1997; revised Dec. 29, 1997; accepted Jan. 2, 1998.
This work was supported by grants from the Sasakawa Health Foundation,
the Nagase Science and Technology Foundation, the Brain Science
Foundation, the Grant-in-Aid for Scientific Research on Priority Areas
(intracellular proteolysis, targeted recombination), the Grant-in-Aid
for Scientific Research from the Ministry of Education, Science,
Sports, and Culture of Japan, CREST, the Japan Science and Technology
Corporation, and the Ministry of Health and Welfare of Japan. We thank
Dr. R. Mulligan for the pPNT vector and Drs. Akiyama, R. Taniuchi, N. Maruyama, M. Ogawara, H. Nakano, and K. Okumura for their assistance
and discussion.
Correspondence should be addressed to Dr. Takuji Shirasawa, Department
of Neurophysiology, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo-173, Japan.
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REFERENCES |
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Abstract
Introduction
