Overexpression of Full-length but Not N-terminal Truncated Isoform of Microtubule-associated Protein (MAP) 1B Accelerates Apoptosis of Cultured Cortical Neurons*

Yoko UchidaDagger

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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

beta -amyloid (Abeta ) is presumed to play a pathogenic role in Alzheimer's disease (AD). However, there is an imperfect correlation between Abeta deposition and neuronal loss or dementia. To clarify neuronal responses to Abeta , Abeta -induced gene expression in cultured cortical neurons was analyzed by differential display followed by Northern blotting. Here we report that nonaggregated or aggregated Abeta induced microtubule-associated protein 1B (MAP1B) mRNA, especially the alternative transcript containing exon 3U, before disruption of the cell membrane by Abeta . 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 Abeta 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The accumulation of beta -amyloid (Abeta )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 Abeta 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 Abeta production (2), and Abeta 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 Abeta may play an important role in the pathogenesis of AD. However, about half of non-demented aged individuals have Abeta plaques in the neocortex (5-7), and transgenic mice expressing mutant human amyloid precursor protein (APP) with V171F or K670N/M671L develop Abeta 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 Abeta induces not only molecules that activate the cell death pathway but also molecules that protect neurons from Abeta toxicity in the neocortex.

To begin to understand the molecular mechanisms of Abeta toxicity and the protective response of neurons against Abeta , we applied the method of RNA differential display to isolate the genes implicated in Abeta toxicity or protective responses to Abeta . The results presented here demonstrate that nonaggregated or aggregated Abeta 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta -mercaptoethanol.

Treatment with Abeta Peptides-- For treatment with nonaggregated Abeta peptide, Abeta -(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 Abeta peptide was added to the 7-DIV cultures at a concentration of 5 µM immediately after preparation. For treatment with aggregated Abeta peptide, Abeta -(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 beta -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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Time Course of Abeta Neurotoxicity-- The neurotoxicity of Abeta -(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 Abeta -(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 Abeta (p < 0.05) (Fig. 1B). After the addition of nonaggregated Abeta 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 Abeta peptide. These results indicate that the decline in metabolic activity induced in neurons by Abeta treatment occurs before the disruption of the plasma membrane and that nonaggregated Abeta is more toxic than aggregated Abeta .


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Fig. 1.   Time course of nonaggregated or aggregated Abeta -(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 Abeta -(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 Abeta -(1-42) Treatment before Neuronal Death-- To identify Abeta -responsive genes by differential display RT-PCR before the disruption of the cell membrane by Abeta , we compared RNA fingerprinting patterns from neurons exposed to Abeta -(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 Abeta . 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 Abeta (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 Abeta -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 Abeta -(1-42) for 3 h. Poly(A)+RNA extracted from cortical neurons treated for 3 h with nonaggregated or aggregated Abeta -(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 Abeta -(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 Abeta -(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 Abeta -(1-42) for 3 h (Fig. 3C). The mRNAs of microtubule-associated proteins other than MAP1B were also analyzed by Northern blotting. However, neither tau , MAP1A, nor beta -tubulin mRNA was not affected by treatment with nonaggregated or aggregated Abeta -(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 Abeta -(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) Abeta -(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, tau , and beta -tubulin in cortical neurons treated with nonaggregated or aggregated Abeta -(1-42). One microgram aliquots of poly(A)+RNA extracted from cortical neurons treated with either nonaggregated or aggregated Abeta -(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 Delta 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 Delta 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 (Delta 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).

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.

To confirm the apoptotic properties of the MAP1B isoform containing the N-terminal 126 amino acid fragment, full-length or shorter isoforms (MAP1B Delta 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 Delta 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 Delta 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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 Abeta 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 Abeta induces the aberrant sprouting (27). If deposition of Abeta or plaque-associated molecules induces the aberrant sprouting before neurodegeneration, it is reasonable to speculate that Abeta or these plaque-associated molecules cause the re-expression of developmentally regulated proteins. In the present study, we demonstrated that nonaggregated or aggregated Abeta induced MAP1B mRNA, especially the alternative transcript-containing exon 3U, suggesting that Abeta 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 Delta 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 tau -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 tau  does (30).

As expected from the aberrant axonal sprouting before neurodegeneration in APP transgenic mice (27), Abeta 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-Abeta 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.

Dagger 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

    ABBREVIATIONS

The abbreviations used are: Abeta , beta -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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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