Overexpression of Full-length but Not N-terminal
Truncated Isoform of Microtubule-associated Protein (MAP) 1B
Accelerates Apoptosis of Cultured Cortical Neurons*
Yoko
Uchida
From the Gene Expression Research Group, Division of Neuroscience
and Brain Function, Tokyo Metropolitan Institute of Gerontology,
35-2 Sakaecho, Itabashiku, Tokyo 173-0015, Japan
Received for publication, October 2, 2002
 |
ABSTRACT |
-amyloid (A
) is presumed to play a
pathogenic role in Alzheimer's disease (AD). However, there is an
imperfect correlation between A
deposition and neuronal loss or
dementia. To clarify neuronal responses to A
, A
-induced gene
expression in cultured cortical neurons was analyzed by differential
display followed by Northern blotting. Here we report that
nonaggregated or aggregated A
induced microtubule-associated protein
1B (MAP1B) mRNA, especially the alternative transcript containing
exon 3U, before disruption of the cell membrane by A
. An alternative
transcript containing exon 3U is translated into an N-terminal
truncated shorter isoform of MAP1B. Transfection experiments reveal
that overexpression of this isoform does not accelerate neurite
outgrowth or apoptosis of cortical neurons. In contrast, overexpression
of MAP1B fragments containing the N-terminal 126 amino acids promoted
neurite outgrowth and neuronal apoptosis. These results suggest that
A
does not induce deleterious full-length MAP1B directly, but
overexpression of full-length MAP1B might act as an effector of cell
death in neurodegenerative disorders related to cytoskeletal abnormalities.
 |
INTRODUCTION |
The accumulation of
-amyloid
(A
)1 plaques and
neurofibrillary tangles and neuronal loss in the neocortex are
hallmarks of Alzheimer's disease (AD). Pathological studies of Down's
syndrome have indicated that deposition of A
throughout the
neocortex is the earliest event among the three lesions seen in the AD
neocortex (1). Moreover, mutations in the three genes associated with familial AD cause increases in A
production (2), and A
has a
toxic effect on cultured neuronal cells via an increase in reactive oxygen species production and/or activation of specific immediate-early genes (3, 4). These observations indicate that A
may play an
important role in the pathogenesis of AD. However, about half of
non-demented aged individuals have A
plaques in the neocortex (5-7), and transgenic mice expressing mutant human amyloid precursor protein (APP) with V171F or K670N/M671L develop A
plaques in the
neocortex progressively with age (8, 9) but do not show neuronal loss
in the neocortex (10, 11). These contradictory findings in
vitro and in vivo suggest that A
induces not only molecules that activate the cell death pathway but also molecules that
protect neurons from A
toxicity in the neocortex.
To begin to understand the molecular mechanisms of A
toxicity and
the protective response of neurons against A
, we applied the method
of RNA differential display to isolate the genes implicated in
A
toxicity or protective responses to A
. The results presented here demonstrate that nonaggregated or aggregated A
induces MAP1B mRNA, especially the alternative transcript containing exon 3U. Transfection experiments of MAP1B isoforms in cultured cortical neurons
indicated that full-length MAP1B but not the alternative MAP1B isoform
resulted in the acceleration of neuronal death.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture--
Cerebral cortices dissected from day E17
embryonic rats were dissociated by incubation with 0.08%
trypsin/0.008% DNase I at 37 °C for 10 min and passed through a
62-µm nylon mesh. The cells (4 × 104 cell/well or
4.5 × 106 cells/dish, respectively) were seeded in
96-well plates or 6-cm dishes, both of which were precoated with
gelatin-polyornithine, and were cultured for 7 days in MEM with 5%
fetal bovine serum and 10 µM
-mercaptoethanol.
Treatment with A
Peptides--
For treatment with
nonaggregated A
peptide, A
-(1-42) (Bachem) was dissolved
at 250 µM in 0.05 N HCl, filtered through a 0.45-µm
membrane filter, diluted with MEM with N2 supplement (MEM-N2), and
neutralized. The nonaggregated A
peptide was added to the 7-DIV
cultures at a concentration of 5 µM immediately after
preparation. For treatment with aggregated A
peptide, A
-(1-42)
was dissolved at 250 µM in 0.05 N HCl,
neutralized, and incubated at 37 °C for 4 days. The aggregated
peptide suspension was diluted with MEM-N2 and added to the 7 DIV
cultures at a concentration of 5 µM. Neuronal viability
was determined by an MTT assay (12) and trypan blue exclusion.
mRNA Differential Display--
Poly(A)+RNA from
cultured rat neurons was isolated using an Amersham Biosciences
Micro mRNA purification kit. Reverse transcription was carried out
using AMV reverse transcriptase XL (Life Sciences) and the three
two-base-anchored oligo(dT) primers (T11), GG, CG, or AA. PCR
amplification was performed with the three oligo(dT) primers in
combination with 24 arbitrary primers (Display Systems Biotech) in the
presence of [32P]dCTP. PCR products were electrophoresed
on Gene Gel Clean (Amersham Biosciences) and exposed to the imaging
plate of a Fuji Bioimage analyzer BAS 2500. cDNAs were eluted from
differentially displayed bands, amplified with the same primer sets
described above, and cloned into a pCR2.1 vector (Invitrogen).
Northern Blot Analysis--
Aliquots of 1 µg of poly
(A)+RNA were denatured, electrophoretically fractionated on
a 1.4% agarose/formaldehyde gel, and transferred to a nylon membrane.
Hybridization was performed in the solution containing cloned cDNA
labeled with [32P]dCTP using a random labeling kit (Roche
Molecular Biochemicals). Radioactivities of the bands were measured
using a Fuji Bioimage analyzer BAS 2500.
cDNA Library Screening--
A 64-bp cDNA fragment
obtained by the differential display method was used to screen 4 × 105 colonies from a SuperScript Rat Neuronal Cell
cDNA Library (Invitrogen). The corresponding DNA was sequenced
using a Taq cycle sequencing kit (Takara) with a
fluorescence autosequencer ABI377.
RT-PCR of MAP1B--
RT-PCR analysis was carried out according
to the methods of Kutschera et al. (1998) (13). Briefly,
first-strand cDNA was synthesized from poly(A)+RNA of
cultured rat neurons using SuperScript II and random hexamers (Invitrogen). PCR was carried out with ELONGase Enzyme Mix
(Invitrogen). For amplification of regular transcripts of rat MAP1B,
the upstream primer was nucleotides 169-190 (accession no.
U52950) (14), which are located in exon 1 of MAP1B. For
amplification of alternative transcripts containing exon 3U (accession
no. AF035827) and 3A (accession no. AF035829), the upstream primers
were nucleotides
98 to
74 and
58 to
34, respectively (13). The
downstream primer was nucleotides 684-660, which are located in exon 5 of MAP1B (accession no. X60370) (15) for all amplifications. PCR
products were electrophoretically fractionated on a 1.4% agarose gel
and transferred to a nylon membrane. A cDNA probe of MAP1B for
Southern hybridization was obtained by RT-PCR using primers corresponding to nucleotides 191-208 (located in exon 1) and 620-602 (located in exon 5). Hybridization was performed in the solution containing cloned cDNA labeled using the [32P[dCTP
random labeling kit.
Construction of MAP1B--
Full-length rat cDNA for MAP1B
was generated by RT-PCR. First-strand cDNA was synthesized using
SuperScript II and poly(A)+RNA from cultured rat neurons.
PCR was carried out with ELONGase Enzyme Mix. Upstream primers were
nucleotides 26-48, 253-274, 1404-1426, 3156-3179, 4042-4063,
5053-5080, and 6181-6204. Downstream primers were nucleotides
656-632, 1663-1641, 4161-4139, 4841-4818, 6280-6259, 7061-7036,
and 7451-7428, respectively (14, 15). Each PCR product was cloned into
pCR2.1 vector (Invitrogen). A cDNA for alternative transcripts
containing exon 3U was cloned by RT-PCR using an upstream primer
located in exon 3U, nucleotides
409 to
386 (13) and a downstream
primer located in exon 4, nucleotides 478-458 (14, 15). The PCR
fragment containing exon 3U was fused to the AatII site of
full-length MAP1B. The nucleotide sequences of all PCR fragments were
analyzed to confirm the authenticity of rat MAP1B cDNA. The
full-length transcript and the alternative transcript starting from
exon 3U of MAP1B were cloned into pCMV-Tag 5 containing a c-Myc epitope (Stratagene).
Transfection--
The constructs were transfected into 6-DIV
cortical neurons with LipofectAMINE 2000 according to the
manufacturer's manual (Invitrogen). For re-plating experiments, cells
were removed from dishes 20 h after transfection with 0.05%
trypsin, washed with MEM/10% FBS three times, and seeded into 6-cm
dishes precoated with gelatin-polyornithine. Re-plated cells were
cultured for 6 h in MEM with 5% fetal bovine serum and 10 µM
-mercaptoethanol. For serum withdrawal experiments,
the culture medium was replaced with MEN-N2 20 h after
transfection, and the cells were cultured for an additional 24 h.
Immunofluorescence--
Cells were incubated with 10 µM Hoechst 33342 in PBS (
) for 10 min, fixed with
ethanol at
20 °C for 10 min, blocked with 2% skim milk for 30 min, and reacted with the primary antibodies for 1 h followed by
1 h of reaction with the secondary antibodies. The primary
antibodies used were polyclonal anti-Myc-tag antibodies (MBL
International) and a monoclonal anti-MAP1B antibody (AA6). Secondary
antibodies were Texas Red-conjugated anti-rabbit IgG (Vector
Laboratories) and fluorescein-conjugated anti-mouse Ig (Amersham
Biosciences). Transfected cells were visualized with an Olympus
epifluorescence microscope. At least 400 transfected cells were
examined for each construct for measuring the neurite length.
Apoptotic neurons were counted in at least 600 transfected cells
for each construct and in each transfection experiment.
Immunoblotting--
Cells were harvested 20 h after
transfection, extracted with radioimmune precipitation assay (RIPA)
buffer containing 2 mM EDTA and protease inhibitors, and
centrifuged 20 min at 14,000 rpm at 4 °C. Cell lysates were analyzed
by SDS-PAGE (a 3-10% acrylamide linear gradient gel). After the
proteins were transferred to Immobilon, a Myc-tag was detected with
anti-Myc-tag antibodies (MBL International) by the enhanced
chemiluminescence method.
 |
RESULTS |
Time Course of A
Neurotoxicity--
The neurotoxicity of
A
-(1-42) to rat cortical neurons was assessed by the MTT reduction
or the trypan blue exclusion. A significant decrease in MTT reduction
was detected after 3 h of either nonaggregated or aggregated
A
-(1-42) treatment (p < 0.001 or p < 0.05, respectively) and continued until at least 48 h of
treatment (Fig. 1A), whereas the neuronal viability assessed by trypan blue exclusion began to
decrease at 6 h of treatment with either nonaggregated or
aggregated A
(p < 0.05) (Fig.
1B). After the addition of nonaggregated A
peptide, the neuronal viability continuously decreased for at least up
to 48 h; however, the decrease in neuronal viability reached a
plateau at 24 h after treatment with aggregated A
peptide. These results indicate that the decline in metabolic activity induced
in neurons by A
treatment occurs before the disruption of the plasma
membrane and that nonaggregated A
is more toxic than aggregated
A
.

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Fig. 1.
Time course of nonaggregated or aggregated
A -(1-42) toxicity in 7-DIV cortical
neurons. Rat cerebral cortical neurons were seeded and cultured
for 7 days as indicated under "Experimental Procedures." 7-DIV
cortical neurons were treated with nonaggregated or aggregated
A -(1-42) (final concentration 5 µM) for an additional
1-48 h. Neuronal viability was assayed by MTT reduction (A)
or by trypan blue exclusion (B). Data are mean ± S.E.
of two independent experiments with four determinations each. Results
are expressed as a percentage of the untreated control value.
|
|
MAP1B Was Induced by Either Nonaggregated or Aggregated
A
-(1-42) Treatment before Neuronal Death--
To identify
A
-responsive genes by differential display RT-PCR before the
disruption of the cell membrane by A
, we compared RNA fingerprinting
patterns from neurons exposed to A
-(1-42) (5 µM) for
3 h with those from control neurons. Most of the bands observed in
this screening using 72 primer pairs showed the same patterns in
control neurons and in neurons treated with nonaggregated or aggregated
A
. A cDNA band obtained with the primer set T11GG and upstream
primer no. 13 (5'-TGGATTGGTC-3') showed an increase in the neurons
treated with either nonaggregated or aggregated A
(Fig.
2A). This band contained a
64-bp cDNA (clone i132). Northern blot analysis using clone i132 as
a probe confirmed the differential expression in A
-treated neurons
(Fig. 2B). By iterative screening of a nerve cell cDNA
library using the clone i132 as a probe, a cDNA (3.572 kb) was
identified as the 3'-untranslated region of MAP1B (accession no.
AF115776).

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Fig. 2.
Differential display screening of rat
cortical neurons treated with nonaggregated or aggregated
A -(1-42) for 3 h.
Poly(A)+RNA extracted from cortical neurons treated for
3 h with nonaggregated or aggregated A -(1-42) (final
concentration 5 µM) was used either for differential
display RT-PCR screening (A) or Northern blot analysis
(B). A, amplified 32P-labeled PCR
products using the primer set T11GG and 5'-TGGATTGGTC-3' were separated
by electrophoresis. The arrow shows the selected band, i132.
B, cDNAs were eluted from the band selected in
panel A, reamplified, and used as probes for
Northern blotting. One microgram of the same poly(A)+RNA
samples were electrophoresed, transblotted to a nylon membrane, and
hybridized with i132 probe and a glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) probe. CNT, control.
|
|
Alternative MAP1B Transcript Containing Exon 3U Was Up-regulated in
Cortical Neurons Treated with Either Nonaggregated or Aggregated
A
-(1-42)--
The MAP1B gene is transcribed into three different
transcripts, i.e. a regular transcript containing exons 1-7
and two alternative transcripts containing either exon 3U or 3A and
3-7 (13). RT-PCR using 5'-specific primers located in exon 1, 3U, and
3A and a common 3'-primer located in exon 5 followed by Southern blot
hybridization using a MAP1B-specific probe showed that only the
transcript containing exon 3U increased in neurons treated with either
nonaggregated or aggregated A
-(1-42) for 3 h (Fig.
3B). Northern blot analysis confirmed the significant induction of the alternative transcript containing exon 3U in cortical neurons treated with nonaggregated or
aggregated A
-(1-42) for 3 h (Fig. 3C). The
mRNAs of microtubule-associated proteins other than MAP1B were also
analyzed by Northern blotting. However, neither
, MAP1A, nor
-tubulin mRNA was not affected by treatment with nonaggregated
or aggregated A
-(1-42) for 3-24 h (Fig. 3C).

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Fig. 3.
An alternative MAP1B transcript containing
exon 3U was up-regulated in cortical neurons treated with either
nonaggregated or aggregated A -(1-42) for
3 h. A, schematic representation of regular and
alternative transcripts of rat MAP1B. Exons are shown by
boxes with the exon number. B, RT-PCR analysis
followed by Southern blot hybridization of three MAP1B transcripts in
cortical neurons treated with nonaggregated (Non) or
aggregated (Agg) A -(1-42) for 3 h. Aliquots of
first-strand cDNA from poly(A)+RNA were subjected to
PCR using the same downstream primer located in exon 5 and the upstream
primers in exon 1, 3U, or 3A (the positions of primers are illustrated
as bars in panel A). Southern blot
hybridization was carried out using an MAP1B-specific probe. The
fragments showed the expected size for the regular transcript
containing exons 1-5 (515 bp), the alternative transcript containing
exons 3U to 5 (435 bp), or the alternative transcript containing exons
3A to 5 (395 bp). CNT, control. C, Northern blot
analysis of regular MAP1B, an alternative MAP1B transcript containing
exon 3U, MAP1A, , and -tubulin in cortical neurons treated with
nonaggregated or aggregated A -(1-42). One microgram aliquots of
poly(A)+RNA extracted from cortical neurons treated with
either nonaggregated or aggregated A -(1-42) for 3, 6, and 24 h
were electrophoresed, transblotted to a nylon membrane, and hybridized
with specific probes. Data are mean ± S.D. of three experiments.
**, p < 0.01; ***, p < 0.001 by
analysis of variance (ANOVA) and the post hoc test compared
with untreated control.
|
|
MAP1B Fragment Containing N-terminal 126 Amino Acids Induced
Apoptosis in Cortical Neurons--
MAP1B is a minor component of
paired helical filaments found in Alzheimer's disease brains (16-19)
and also of cortical Lewy bodies in Parkinson's disease (20, 21). It
is not known which isoforms, full-length MAP1B or shorter isoforms
(MAP1B
126), are involved in the formation of paired helical
filaments or Lewy bodies. To investigate which isoforms of MAP1B are
responsible for the degeneration of neurons, we examined the effects of
overexpression of MAP1B fragments on neurite sprouting and neuronal
apoptosis. Because the indistinguishable electrophoretic mobility
between full-length MAP1B (MAP1B-(1-2459)) and MAP1B
126
(MAP1B-(127-2459)) makes it difficult to examine the expression of
each MAP1B isoform in transfected neurons by Western blotting, MAP1B
fragments MAP1B-(1-1367) and MAP1B-(127-1367), both of which were
tagged with c-Myc epitope, were generated and expressed in 6-DIV
cortical neurons. Fragments MAP1B-(1-1367) and MAP1B-(127-1367) were
detectable with anti-Myc antibodies and an authentic MAP1B antibody,
AA6. The expression of exogenous MAP1B proteins was confirmed by
Western blotting (Fig. 4B) and
immunocytochemistry (Fig. 4, C and D) using an
antibody against c-Myc. As shown, both the construct encoding amino
acids 1-1367 and the one encoding 127-1367 gave rise to a protein
band with a different size. Both proteins expressed in cortical neurons were larger than those deduced from the predicted amino acid sequence (152 or 138 kDa, respectively), as was full-length MAP1B (280-300 kDa
in SDS-PAGE but 269 kDa deduced from the predicted amino acid sequence). Immunofluorescence double staining for c-Myc and MAP1B indicated that transfected neurons expressed both MAP1B fragments with
distribution patterns similar to that of endogenous MAP1B (Fig.
4C). The transfection efficiencies of constructs
MAP1B-(1-1367) or MAP 1B-(127-1367) were 4.5% or 2.7%,
respectively, indicating that the lower level of fragment
MAP1B-(127-1367) expression in Western blot reflects, in part, the
lower transfection efficiency of construct MAP1B-(127-1367) in
cortical neurons.

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Fig. 4.
Distribution of transfected MAP1B
fragments. A, schematic representation of MAP1B
fragments and constructs used in this study. Depicted are the
full-length rat MAP1B polyprotein precursor (Full-length,
amino acids 1-2459), the shorter isoform encoded by the cDNA
construct corresponding to exons 3U, 3, 4, 5, 6, and 7, whose
translation started at ATG in exon 4 ( 126, amino acids
127-2459), the MAP1B fragment encoded by the cDNA construct
corresponding to exons 1 to 5 (1B(1-1367), amino acids
1-1367), and the MAP1B fragment encoded by the cDNA construct
corresponding to exons 3U to 5 (1B(127-1367), amino acids
127-1367). The Myc-tag was attached to the C termini of all proteins
and fragments. B, Western blot of MAP1B fragments expressed
in 6-DIV cortical neurons. Cell lysates of cortical neurons transfected
with the indicated constructs were fractionated by SDS-PAGE on a
3-10% linear gradient gel and analyzed by immunoblotting with Myc-tag
antibodies. C and D, distribution of MAP1B
fragments expressed in 6-DIV cortical neurons. Cortical neurons
transfected with the constructs MAP1B-(1-1367) (1B(1-1367),
panel C) or MAP1B-(127-1367) (1B(127-1367), panel
D) were double-stained with polyclonal anti-Myc-tag
antibodies (C and D, left) and
monoclonal anti-MAP1B, AA6 (C and D,
right).
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|
To examine the effect of overexpression of MAP1B fragments on neurite
extension, cortical neurons transfected with constructs MAP1B-(1-1367)
or MAP1B-(127-1367) were enzymatically removed from dishes, re-plated
onto gelatin-polyornithine-coated dishes, and cultured for
6 h. As shown in Fig. 5A,
overexpression of fragment MAP1B-(1-1367) in cortical neurons shifted
the peak of neurite length to a longer range. The average neurite
lengths of MAP1B-(1-1367)-expressing (63.1 ± 1.3 µm) and
MAP1B(127-1367)-expressing neurons (54.9 ± 1.0 µm) were
significantly different (p < 0.0001). To examine the
effect of overexpression of MAP1B fragments on neuronal apoptosis after serum withdrawal, cortical neurons expressing MAP1B-(1-1367) or
MAP1B-(127-1367) and showing DNA fragmentation were visualized by
immunostaining with anti-Myc antibodies and Hoechst 33342, and the
percentage of apoptotic neurons relative to the total number of
transfected neurons was determined. Fig. 5B shows that the
overexpression of MAP1B-(1-1367) in neurons induced a significantly higher level of apoptosis than that of MAP1B-(127-1367) or
untransfection.

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Fig. 5.
MAP1B fragment MAP1B-(1-1367) but not
MAP1B-(127-1367) promoted neurite outgrowth and apoptosis in cortical
neurons. Six-DIV rat cortical neurons were transfected for 20 h with constructs MAP1B-(1-1367) or MAP1B-(127-1367). A,
cells were removed from dishes 20 h after transfection, re-plated
on gelatin-polyornithine dishes, and cultured for 6 h. Neurite
length was measured after immunostaining for the Myc-tag in at least
400 transfected cells for each construct in two independent
experiments. B, the culture medium was replaced with
serum-free medium 20 h after transfection, and cells were cultured
for an additional 16 h. The number of transfected neurons
undergoing apoptosis was determined after immunostaining for the
Myc-tag and Hoechst 33342 staining in at least 100 transfected cells
for each construct in each determination. Data are mean ± S.E. of
two independent experiments with three determinations each. Results are
expressed as the percentage of apoptotic cells among Myc-positive
cells. *, p < 0.05 by analysis of variance (ANOVA) and
the post hoc test compared with untransfected
cells.
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To confirm the apoptotic properties of the MAP1B isoform containing the
N-terminal 126 amino acid fragment, full-length or shorter isoforms
(MAP1B
126), both of which were tagged with the c-Myc epitope, were
expressed in 6-DIV cortical neurons, and their effects on neuronal
apoptosis were assessed after serum withdrawal. Fig.
6 shows that neurons expressing
full-length MAP1B were more sensitive to serum withdrawal than those
expressing MAP1B
126 or untransfected neurons. These results
indicate that overexpression of the MAP1B fragment containing the
N-terminal 126 amino acids in cortical neurons may make neurons
vulnerable to apoptotic signals.

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Fig. 6.
Full-length but not N-terminal truncated form
of MAP1B induced apoptosis in cortical neurons. Six-DIV rat
cortical neurons were transfected with the constructs of full-length or
N-terminal truncated 126 of MAP1B. Culture medium was replaced with
serum-free medium 20 h after transfection, and cells were cultured
for an additional 8, 14, or 24 h. The number of transfected
neurons undergoing apoptosis was determined after immunostaining for
the Myc-tag and Hoechst 33342 staining in at least 100 transfected
cells for each construct in each determination. Data are mean ± S.E. of two independent experiments with three determinations each.
Results are expressed as the percentage of apoptotic cells among
Myc-positive cells. ***, p < 0.001 by analysis of
variance (ANOVA) and the post hoc test compared
with untransfected cells.
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 |
DISCUSSION |
MAP1B is expressed abundantly in the fetal or neonatal brain
(22-24) but negligibly in the adult brain except in areas with greater
plasticity potential. MAP1B is highly associated with neurofibrillary
tangles and senile plaque neurites in Alzheimer's disease (16-19).
The re-expression of developmentally regulated proteins such as MAP1B
and CRMP2 (25) in the AD neurons may contribute to the aberrant
neuritic sprouting process in the AD brain (26). Two different possible
explanations for this are that the aberrant sprouting may be a neuronal
response to neurodegeneration or activated glial cells at the end stage
of the disease or that the aberrant sprouting may be induced by the
deposition of A
or plaque-associated molecules at an early stage
before neurodegeneration. The aberrant axonal growth in the vicinity of
amyloid plaques in APP-transgenic mice, which develop amyloid plaques
but not neuronal loss, suggests that deposition of A
induces the
aberrant sprouting (27). If deposition of A
or plaque-associated
molecules induces the aberrant sprouting before neurodegeneration, it
is reasonable to speculate that A
or these plaque-associated
molecules cause the re-expression of developmentally regulated
proteins. In the present study, we demonstrated that nonaggregated or
aggregated A
induced MAP1B mRNA, especially the alternative
transcript-containing exon 3U, suggesting that A
may lead to
re-expression of developmentally regulated MAP1B in neurons. Whether
the MAP1B isoform(s) contained in neurofibrillary tangles and senile
plaque neurites is/are the full-length form or the alternative isoforms
still remains unknown. RT-PCR analysis of MAP1B transcripts using human
brain poly(A)+RNA did not reveal which transcript was
up-regulated in the AD brain because of the low quality of the
postmortem poly(A)+RNA for analysis of the 5'-end of the cDNA.
The next issue addressed here was whether the overexpression of
developmentally regulated protein MAP1B induces degeneration of
neurons. We demonstrated that overexpression of full-length MAP1B but
not of the alternative MAP1B isoform (MAP1B
126) in cultured
cortical neurons resulted in good growth of neurites and
acceleration of neuronal death. It is unlikely that acceleration of
neuronal death in MAP1B-overexpressing neurons is due to a high
concentration of MAP1B in neurons. The exogenous MAP1B concentrations in cortical neurons transfected with different MAP1B isoforms were
calculated from the transfection efficiency and level of the MAP1B
protein as determined by Western blotting. The exogenous MAP1B
concentration in neurons expressing fragment MAP1B-(1-1367) was
3.5-fold higher than that in neurons expressing fragment
MAP1B-(127-1367). The immunofluorescence intensity of the MAP1B
epitope as recognized by monoclonal antibody AA6 in neurons expressing
fragment MAP1B-(1-1367) was almost similar to that in neurons
expressing fragment MAP1B-(127-1367). Thus, the susceptibility of
MAP1B-expressing neurons to death may be an isoform-specific feature;
full-length MAP1B may promote neurite sprouting and neuronal death, but
the alternative isoform may not be deleterious. In agreement with our
finding that the alternative isoform of MAP1B does not accelerate
neuronal death, a recent report indicated that up-regulation of the
MAP1B alternative isoform in heterozygotes of MAP1B-deficient mice does
not cause any overt abnormalities in the nervous system (28). The
isoform-specific deleteriousness of MAP1B appeared to be analogous to
the expression of certain
-isoforms in specific tauopaties (29).
However, it is not known whether overexpression of full-length MAP1B
might promote neuronal death via impairing organelle transport as
does (30).
As expected from the aberrant axonal sprouting before neurodegeneration
in APP transgenic mice (27), A
indeed induced a developmentally
regulated protein, an alternative isoform of MAP1B. However, this
isoform of MAP1B did not accelerate neuronal death when it was
overexpressed in cultured cortical neurons. It should not be ruled out
that non-A
components of plaque amyloid, e.g. heparan
sulfate proteoglycan, apoprotein E, agrin, and CLAC-P/collagen type XXV
(31-34), might be responsible for inducing full-length MAP1B. Recent
reports suggested that MAP1B may play a role in the pathogenesis of
neurodegenerative disorders related to cytoskeletal abnormalities. The
high level of MAP1B in Lewy bodies indicates that overexpression of
MAP1B might be involved in the formation of Lewy bodies (21). Mutations
of gigaxonin induce giant axonal neuropathy via loss of gigaxonin-MAP1B
light chain interactions (35). MAP1B, whose isoforms are not clearly
defined, is up-regulated in the immediate-early phase of apoptosis in
cerebellar granule neurons deprived of potassium and serum (36). These
observations suggest that common cell death signal(s) among
neurodegenerative disorders related to cytoskeletal abnormalities might
stimulate the expression of full-length MAP1B, which might act as an
effector of cell death (37). Further studies to identify molecules that induce full-length MAP1B should contribute to our understanding of the
role of MAP1B in neurodegeneration.
 |
ACKNOWLEDGEMENT |
I thank S. Nakano for excellent assistance in
Western blotting.
 |
FOOTNOTES |
*
This work was supported in part by Grants-in-Aid for
Scientific Research (C) from the Ministry of Education, Science, and Culture, Japan.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 813-3964-3241 ext. 3050; Fax: 813-3579-4776; E-mail: uchiday@tmig.or.jp.
Published, JBC Papers in Press, October 9, 2002, DOI 10.1074/jbc.M210091200
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ABBREVIATIONS |
The abbreviations used are:
A
,
-amyloid;
AD, Alzheimer's disease;
MAP1B, microtubule associated protein 1B;
APP, amyloid precursor protein;
MEM, minimum essential medium;
DIV, days in vitro;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;
RT, reverse transcriptase.
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REFERENCES |
1.
|
Wisniewski, K. E.,
Wisniewski, H. M.,
and Wen, G. Y.
(1985)
Ann. Neurol.
17,
278-282[Medline]
[Order article via Infotrieve]
|
2.
|
Hardy, J.
(1997)
Trends Neurosci.
20,
154-159[CrossRef][Medline]
[Order article via Infotrieve]
|
3.
|
Small, D. H.,
Mok, S. S.,
and Bornstein, J. C.
(2001)
Nat. Rev. Neurosci.
2,
595-598[CrossRef][Medline]
[Order article via Infotrieve]
|
4.
|
Estus, S.,
Tucker, H. M.,
van Rooyen, C.,
Wright, S.,
Brigham, E. F.,
Wogulis, M.,
and Rydel, R. E.
(1997)
J. Neurosci.
17,
7736-7745[Abstract/Free Full Text]
|
5.
|
Armstrong, R. A.
(1994)
Neurosci. Lett.
178,
59-62[Medline]
[Order article via Infotrieve]
|
6.
|
Giannakopoulos, P.,
Hof, P. R.,
Mottier, S.,
Michel, J. P.,
and Bouras, C.
(1994)
Acta Neuropathol.
87,
456-468[CrossRef][Medline]
[Order article via Infotrieve]
|
7.
|
Yamaguchi, H.,
Sugihara, S.,
Ogawa, A.,
Oshima, N.,
and Ihara, Y.
(2001)
J. Neuropathol. Exp. Neurol.
60,
731-739[Medline]
[Order article via Infotrieve]
|
8.
|
Games, D.,
Adams, D.,
Alessandrini, R.,
Barbour, R.,
Berthelette, P.,
Blackwell, C.,
Carr, T.,
Clemens, J.,
Donaldson, T.,
Gillespie, F.,
Guido, T.,
Hagopian, S.,
Johnson-Wood, K.,
Khan, K.,
Lee, M.,
Leibowitz, P.,
Lieberburg, I.,
Little, S.,
Masliah, E.,
McConlogue, L.,
Montoya-Zavala, M.,
Mucke, L.,
Paganini, L.,
and Penniman, E.
(1995)
Nature
373,
523-527[CrossRef][Medline]
[Order article via Infotrieve]
|
9.
|
Hsiao, K.,
Chapman, P.,
Nilsen, S.,
Eckman, C.,
Harigaya, Y.,
Younkin, S.,
Yang, F.,
and Cole, G.
(1996)
Science
274,
99-102[Abstract/Free Full Text]
|
10.
|
Irizarry, M. C.,
McNamara, M.,
Fedorchak, K.,
Hsiao, K.,
and Hyman, B. T.
(1997)
J. Neuropathol. Exp. Neurol.
56,
965-973[Medline]
[Order article via Infotrieve]
|
11.
|
Irizarry, M. C.,
Soriano, F.,
McNamara, M.,
Page, K. J.,
Schenk, D.,
Games, D.,
and Hyman, B. T.
(1997)
J. Neurosci.
17,
7053-7059[Abstract/Free Full Text]
|
12.
|
Manthorpe, M.,
Fagnani, R.,
Skaper, S. D.,
and Varon, S.
(1986)
Brain Res.
390,
191-198[Medline]
[Order article via Infotrieve]
|
13.
|
Kutschera, W.,
Zauner, W.,
Wiche, G.,
and Probst, F.
(1998)
Genomics
49,
430-436[CrossRef][Medline]
[Order article via Infotrieve]
|
14.
|
Liu, D.,
and Fischer, I.
(1996)
Gene
172,
307-308
|
15.
|
Zauner, W.,
Kratz, J.,
Staunton, J.,
Feick, P.,
and Wiche, G.
(1992)
Eur. J. Cell Biol.
57,
66-74[Medline]
[Order article via Infotrieve]
|
16.
|
Hasegawa, M.,
Arai, T.,
and Ihara, Y.
(1990)
Neuron
4,
909-918[Medline]
[Order article via Infotrieve]
|
17.
|
Geddes, J. W.,
Lundgren, K.,
and Kim, Y. K.
(1991)
J. Neurosci. Res.
30,
183-191[Medline]
[Order article via Infotrieve]
|
18.
|
Takahashi, H.,
Hirokawa, K.,
Ando, S.,
and Obata, K.
(1991)
Acta Neuropathol.
81,
626-631[Medline]
[Order article via Infotrieve]
|
19.
|
Ulloa, L.,
Montejo de Garcini, E.,
Gomez-Ramos, P.,
Moran, M. A.,
and Avila, J.
(1994)
Brain Res. Brain Res. Rev.
26,
113-122
|
20.
|
Gai, W. P.,
Blumbergs, P. C.,
and Blessing, W. W.
(1996)
Acta Neuropathol.
91,
78-81[CrossRef][Medline]
[Order article via Infotrieve]
|
21.
|
Jensen, P. H.,
Islam, K.,
Kenney, J.,
Nielsen, M. S.,
Power, J.,
and Gai, W. P.
(2000)
J. Biol. Chem.
275,
21500-21507[Abstract/Free Full Text]
|
22.
|
Safaei, R.,
and Fischer, I.
(1989)
J. Neurochem.
52,
1871-1879[Medline]
[Order article via Infotrieve]
|
23.
|
Schoenfeld, T. A.,
McKerracher, L.,
Obar, R.,
and Vallee, R. B.
(1989)
J. Neurosci.
9,
1712-1730[Abstract]
|
24.
|
Garner, C. C.,
Garner, A.,
Huber, G.,
Kozak, C.,
and Matus, A.
(1990)
J. Neurochem.
55,
146-154[Medline]
[Order article via Infotrieve]
|
25.
|
Yoshida, H.,
Watanabe, A.,
and Ihara, Y.
(1998)
J. Biol. Chem.
273,
9761-9768[Abstract/Free Full Text]
|
26.
|
Arendt, T.
(2001)
Neuroscience
102,
723-765[CrossRef][Medline]
[Order article via Infotrieve]
|
27.
|
Phinney, A. L.,
Deller, T.,
Stalder, M.,
Calhoun, M. E.,
Frotscher, M.,
Sommer, B.,
Staufenbiel, M.,
and Jucker, M.
(1999)
J. Neurosci.
19,
8552-8559[Abstract/Free Full Text]
|
28.
|
Gonzalez-Billault, C.,
Demandt, E.,
Wandosell, F.,
Torres, M.,
Bonaldo, P.,
Stoykova, A.,
Chowdhury, K.,
Gruss, P.,
Avila, J.,
and Sanchez, M. P.
(2000)
Mol. Cell. Neurosci.
16,
408-421[CrossRef][Medline]
[Order article via Infotrieve]
|
29.
|
Goedert, M.,
Spillantini, M. G.,
and Davies, S. W.
(1998)
Curr. Opin. Neurobiol.
8,
619-632[CrossRef][Medline]
[Order article via Infotrieve]
|
30.
|
Ebneth, A.,
Godemann, R.,
Stamer, K.,
Illenberger, S.,
Trinczek, B.,
and Mandelkow, E.
(1998)
J. Cell Biol.
143,
777-794[Abstract/Free Full Text]
|
31.
|
Snow, A. D.,
Mar,
Nochlin, D.,
Kinata, K.,
Kato, M.,
Suzuki, S.,
Hassell, J.,
and Wright, T. N.
(1988)
Am. J. Pathol.
133,
456-463[Abstract]
|
32.
|
Namba, Y.,
Tomonaga, M.,
Kawasaki, H.,
Otomo, E.,
and Ikeda, K.
(1991)
Brain Res.
541,
163-166[CrossRef][Medline]
[Order article via Infotrieve]
|
33.
|
Donahue, J. E.,
Berzin, T. M.,
Rafii, M. S.,
Glass, D. J.,
Yancopoulos, G. D.,
Fallon, J. R.,
and Stopa, E. G.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
6468-6472[Abstract/Free Full Text]
|
34.
|
Hashimoto, T.,
Wakabayashi, T.,
Watanabe, A.,
Kowa, H.,
Hosoda, R.,
Nakamura, A.,
Kanazawa, I.,
Arai, T.,
Takio, K.,
Mann, D. M.,
and Iwatsubo, T.
(2002)
EMBO J.
21,
1524-1534[Abstract/Free Full Text]
|
35.
|
Ding, J.,
Liu, J.-J.,
Kowal, A. S.,
Nardine, T.,
Bhattacharya, P.,
Lee, A.,
and Yang, Y.
(2002)
J. Cell Biol.
158,
427-433[Abstract/Free Full Text]
|
36.
|
Chiang, L. W.,
Grenier, J. M.,
Ettwiller, L.,
Jenkins, L. P.,
Ficenec, D.,
Martin, J.,
Jin, F.,
DiStefano, P. S.,
and Wood, A.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
2814-2819[Abstract/Free Full Text]
|
37.
|
Yuan, J.,
and Yankner, B. A.
(1999)
Nat. Cell Biol.
1,
E44-45[CrossRef][Medline]
[Order article via Infotrieve]
|
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