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

(cid:1) -amyloid (A (cid:1) ) is presumed to play a pathogenic role in Alzheimer’s disease (AD). However, there is an imper-fect correlation between A (cid:1) deposition and neuronal loss or dementia. To clarify neuronal responses to A (cid:1) , A (cid:1) -induced gene expression in cultured cortical neurons was analyzed by differential display followed by Northern blotting. Here we report that nonaggregated or aggregated A (cid:1) induced microtubule-associated protein 1B (MAP1B) mRNA, especially the alternative transcript containing exon 3U, before disruption of the cell membrane by A (cid:1) . 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 (cid:1) 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. The accumulation of (cid:1) -amyloid (A (cid:1) ) 1 plaques and neurofibrillary 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 [ 32 P]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 (cid:2) g of poly (A) (cid:2) RNA were denatured, electrophoretically fractionated on a 1.4% agarose/formal-dehyde gel, and transferred to a nylon membrane. Hybridization was performed in the solution containing cloned cDNA labeled with [ 32 P]dCTP using a random labeling kit (Roche Molecular Biochemicals). Radioactivities of the bands were measured using a Fuji Bioimage analyzer BAS 2500.

␤-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 fulllength MAP1B directly, but overexpression of fulllength MAP1B might act as an effector of cell death in neurodegenerative disorders related to cytoskeletal abnormalities.
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)(6)(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-m nylon mesh. The cells (4 ϫ 10 4 cell/well or 4.5 ϫ 10 6 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 M ␤-mercaptoethanol.
Treatment with A␤ Peptides-For treatment with nonaggregated A␤ peptide, A␤-(1-42) (Bachem) was dissolved at 250 M in 0.05 N HCl, filtered through a 0.45-m 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 M immediately after preparation. For treatment with aggregated A␤ peptide, A␤-(1-42) was dissolved at 250 M 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 M. Neuronal viability was determined by an MTT assay (12) and trypan blue exclusion.
mRNA Differential Display-Poly(A) ϩ 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 [ 32 P]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 g of poly (A) ϩ 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 [ 32 P]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 ϫ 10 5 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) ϩ 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 Ϫ98 to Ϫ74 and Ϫ58 to Ϫ34, 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 [ 32 P[dCTP random labeling kit.
Construction of MAP1B-Full-length rat cDNA for MAP1B was generated by RT-PCR. First-strand cDNA was synthesized using Super-Script II and poly(A) ϩ RNA from cultured rat neurons. PCR was carried out with ELONGase Enzyme Mix. Upstream primers were nucleotides 26 - (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 Ϫ409 to Ϫ386 (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 M ␤-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 M Hoechst 33342 in PBS (Ϫ) for 10 min, fixed with ethanol at Ϫ20°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 antirabbit 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 Ͻ 0.001 or p Ͻ 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 Ͻ 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␤.
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 M) 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 (5Ј-TGGAT-TGGTC-3Ј) showed an increase in the neurons treated with either nonaggregated or aggregated A␤ ( Fig. 2A). This band Full-length MAP1B Overexpression Accelerates Neuronal Death 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 3Ј-untranslated region of MAP1B (accession no. AF115776).
Alternative MAP1B Transcript Containing Exon 3U Was Upregulated 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 5Ј-specific primers located in exon 1, 3U, and 3A and a common 3Ј-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).  (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.
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. DISCUSSION MAP1B is expressed abundantly in the fetal or neonatal brain (22)(23)(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-trans- genic 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 plaqueassociated 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 se-nile 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) ϩ RNA did not reveal which transcript was up-regulated in the AD brain because of the low quality of the postmortem poly(A) ϩ RNA for analysis of the 5Ј-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 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)(32)(33)(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.