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Keywords:

  • brain-derived neurotrophic factor;
  • gene expression;
  • human;
  • mRNA;
  • RT-PCR

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Human post-mortem brain tissue
  5. RNA isolation
  6. Primers for BDNF transcripts and β-actin
  7. RT-PCR
  8. Isolation and sequencing of RT-PCR products
  9. Quantitative and statistical analysis
  10. Results
  11. Samples
  12. New alternative splice site in transcript 4I
  13. β-Actin control
  14. Transcripts 1, 2 and 3 are decreased in AD parietal cortex
  15. Discussion
  16. Acknowledgements
  17. References

Brain-derived neurotrophic factor (BDNF) supports hippocampal, cortical and basal forebrain cholinergic neurons, which lose function in Alzheimer's disease. In Alzheimer's tissues such as hippocampus and parietal cortex, brain- derived neurotrophic factor mRNA is decreased three- to four-fold compared with controls. However, the molecular mechanism of the down-regulation of BDNF in Alzheimer's disease is unknown. The human brain-derived neurotrophic factor gene has multiple promoters governing six non-coding upstream exons that are spliced to one downstream coding exon, leading to six different transcripts. Here we report an alternate human splice variant within exon 4I for a total of seven transcripts. Previous brain-derived neurotrophic factor mRNA measurements in Alzheimer's disease tissue were done using the downstream coding exon present in all transcripts. Using RT-PCR primers specific for each upstream exon, we observe a significant decrease in three human brain-derived neurotrophic factor mRNA transcripts in Alzheimer's disease samples compared with controls. Transcripts 1 and 3 each exhibit a two-fold decrease, and transcript 2 shows a five-fold decrease. There are no significant differences between control and Alzheimer's disease samples for the other transcripts, including the new splice variant. In rat, both transcripts 1 and 3 are regulated through the transcription factor cAMP response element binding protein, whose phosphorylation is decreased in the Alzheimer's disease brain. This could lead to specific down-regulation of the brain-derivedneurotrophic factor transcripts shown here.

Abbreviations used
AD

Alzheimer's disease

BDNF

brain-derived neurotrophic factor

CRE

cAMP response element

CREB

cAMP response element binding protein.

Brain-derived neurotrophic factor (BDNF) is highly expressed and is distributed widely throughout the CNS, specifically in the hippocampal formation, cerebral cortex, and amygdaloid complex (Ernfors et al. 1990; Hofer et al. 1990; Phillips et al. 1990; Wetmore et al. 1990). BDNF promotes the survival and function of hippocampal and cortical neurons (Ghosh et al. 1994; Lindholm et al. 1996; Lowenstein and Arsenault 1996), cholinergic neurons (Alderson et al. 1990; Knusel et al. 1991) and nigral dopaminergic neurons (Hyman et al. 1991; Knusel et al. 1991). BDNF is also important for synaptic transmission and the excitatory properties of these neurons (Patterson et al. 1992; Castren et al. 1993; Dragunow et al. 1993; Kang and Schuman 1995; Scharfman 1997; Osehobo et al. 1999; McLean et al. 2000).

Basal forebrain cholinergic, cortical, and hippocampal neurons lose function and synaptic connectivity in Alzheimer's disease (AD) (Coyle et al. 1983; Whitehouse et al. 1982; Cuello and Sofroniew 1984; Etienne et al. 1986; Hefti and Weiner 1986; Mann 1991). This may occur because of a deficit in BDNF in the AD brain. A 3–4-fold reduction in BDNF mRNA has been amply documented in the hippocampus and parietal cortex (Phillips et al. 1991; Holsinger et al. 2000). Protein levels of BDNF have been shown to decrease in Alzheimer's disease entorhinal cortex, hippocampus and temporal, frontal and parietal cortex (Narisawa-Saito et al. 1996; Connor et al. 1997; Ferrer et al. 1999; Hock et al. 2000). However, the transcriptional regulation of the human BDNF gene has not been studied, and so the mechanism of the decrease in BDNF levels in the AD brain is not well understood.

In the rat, the BDNF gene has been shown to have four 5′ exons and one 3′ exon (Timmusk et al. 1993). The four 5′ exons each have upstream promoters and each is individually spliced to the 3′ exon (encoding the mature protein) to give four different transcripts. Downstream of the 3′ exon there are two polyadenylation sites, which give two different length transcripts for each upstream exon, totaling eight splice variants (Timmusk et al. 1993). The presence of multiple promoters in the BDNF gene allow for differential mechanisms of activation and tissue-specific expression in the CNS (Falkenberg et al. 1992; Metsis et al. 1993; Kokaia et al. 1994; Timmusk et al. 1995).

The human BDNF gene is structurally similar to the rat gene (Maisonpierre et al. 1991). The human BDNF gene contains two additional non-coding exons compared with rat (4I and 5U), but only one polyadenylation site is present downstream from the 3′ coding exon (Aoyama et al. 2001). The six upstream exons in the human BDNF gene give rise to six transcripts, although the additional exons produce transcripts by differential splicing, not additional promoters (Aoyama et al. 2001). Previous measurements documenting decreased BDNF mRNA in the AD brain targeted the coding exon (exon5) present in all transcripts and therefore examined total BDNF mRNA levels. In this study, we used RT-PCR with upstream primers specific for each exon to determine which of the transcripts is responsible for the reduced BDNF mRNA in AD brain.

Human post-mortem brain tissue

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Human post-mortem brain tissue
  5. RNA isolation
  6. Primers for BDNF transcripts and β-actin
  7. RT-PCR
  8. Isolation and sequencing of RT-PCR products
  9. Quantitative and statistical analysis
  10. Results
  11. Samples
  12. New alternative splice site in transcript 4I
  13. β-Actin control
  14. Transcripts 1, 2 and 3 are decreased in AD parietal cortex
  15. Discussion
  16. Acknowledgements
  17. References

Parietal cortex tissue samples from normal, neurologically unimpaired subjects (n = 12; six females, six males) and from subjects with AD (n = 12; six females and six males) were provided by the Institute for Brain Aging and Dementia Tissue Repository at the University of California, Irvine. A diagnosis of AD was confirmed by pathological and clinical criteria (McKhann et al. 1984; Khatchaturian 1985). Control and AD samples were matched for age and gender. Tissue was frozen at autopsy and stored at −80°C until use.

RNA isolation

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Human post-mortem brain tissue
  5. RNA isolation
  6. Primers for BDNF transcripts and β-actin
  7. RT-PCR
  8. Isolation and sequencing of RT-PCR products
  9. Quantitative and statistical analysis
  10. Results
  11. Samples
  12. New alternative splice site in transcript 4I
  13. β-Actin control
  14. Transcripts 1, 2 and 3 are decreased in AD parietal cortex
  15. Discussion
  16. Acknowledgements
  17. References

Total cellular RNA was purified from parietal cortex samples using TRIzolTM Reagent (Gibco BRL, Burlington, Ontario, Canada) following the manufacturer's protocol. Samples exhibiting an absorbance ratio (260/280) greater than or equal to 1.7 and exhibiting strong 28S and 18S ribosomal RNA bands on 0.01-g/mL agarose gels were used for further analysis.

Primers for BDNF transcripts and β-actin

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Human post-mortem brain tissue
  5. RNA isolation
  6. Primers for BDNF transcripts and β-actin
  7. RT-PCR
  8. Isolation and sequencing of RT-PCR products
  9. Quantitative and statistical analysis
  10. Results
  11. Samples
  12. New alternative splice site in transcript 4I
  13. β-Actin control
  14. Transcripts 1, 2 and 3 are decreased in AD parietal cortex
  15. Discussion
  16. Acknowledgements
  17. References

Human BDNF primer sequences were kindly provided by DrMineyoshi Aoyama, Department of Bioregulation Research, Nagoya City University Medical School, Mizuho-ku, Nagoya, Japan. β-Actin primer sequences were previously described (St Amand et al. 1996). Primers were synthesized at the Central Facility of the Institute for Molecular Biology and Biotechnology (MOBIX) at McMaster University.

RT-PCR

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Human post-mortem brain tissue
  5. RNA isolation
  6. Primers for BDNF transcripts and β-actin
  7. RT-PCR
  8. Isolation and sequencing of RT-PCR products
  9. Quantitative and statistical analysis
  10. Results
  11. Samples
  12. New alternative splice site in transcript 4I
  13. β-Actin control
  14. Transcripts 1, 2 and 3 are decreased in AD parietal cortex
  15. Discussion
  16. Acknowledgements
  17. References

For determination of transcripts 2 and 5U, RNA samples were treated with DNaseI (1.0 µL/10 µg total RNA; Ambion, Austin, TX, USA) at 37°C for 30 min, followed by 2 µL of DNase Inactivation reagent. For all other transcripts, RNA samples did not undergo DNase treatment. Ten micrograms total RNA from human parietal cortex was reverse transcribed into cDNA using the GeneAmp® RNA PCR kit (Perkin Elmer, Norwalk, CT, USA). PCR was performed in the GeneAmp PCR system 2400 using 5 µL aliquots of the reverse transcriptase reaction mixture with 0.35 µm each of the 3′ and 5′ primers, 16.8 µCi of 33P-dCTP, and 2.5 U AmpliTaq Gold (Perkin Elmer). Optimization was performed for all primer sets to determine an optimal cycle number within the logarithmic phase of amplification. Cycle optimization for transcripts are as follows, transcript 1, 35 cycles, transcript 2, 34 cycles, transcript 3, 35 cycles, transcript 4, 32 cycles, transcript 4I, 38 cycles, transcript 5U, 35 cycles, and β-actin, 22 cycles. The amplification profile included an initial activation of the Taq polymerase for 12 min at 95°C, denaturation for 30 s at 94°C followed by annealing at 58°C for 30 s, extension at 72°C for 45 s, and a final extension at 72°C for 7 min. For β-actin, annealing was at 64°C for 30 s and extension at 72°C for 1 min. Four to five independent RT-PCRs were performed for each primer pair.

Isolation and sequencing of RT-PCR products

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Human post-mortem brain tissue
  5. RNA isolation
  6. Primers for BDNF transcripts and β-actin
  7. RT-PCR
  8. Isolation and sequencing of RT-PCR products
  9. Quantitative and statistical analysis
  10. Results
  11. Samples
  12. New alternative splice site in transcript 4I
  13. β-Actin control
  14. Transcripts 1, 2 and 3 are decreased in AD parietal cortex
  15. Discussion
  16. Acknowledgements
  17. References

Amplified RT-PCR product from transcript 4I gave two bands upon electrophoresis in a 0.018-g/mL agarose gel in the presence of ethidium bromide. Single bands were cut from the gel, and DNA was isolated using QIAquickTM Gel Extraction Kit (Qiagen, Mississauga, ON, Canada). The isolated bands were sequenced in both directions, using transcript 4I PCR primers, by the Central Facility of the Institute for Molecular Biology and Biotechnology (MOBIX) at McMaster University.

Quantitative and statistical analysis

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Human post-mortem brain tissue
  5. RNA isolation
  6. Primers for BDNF transcripts and β-actin
  7. RT-PCR
  8. Isolation and sequencing of RT-PCR products
  9. Quantitative and statistical analysis
  10. Results
  11. Samples
  12. New alternative splice site in transcript 4I
  13. β-Actin control
  14. Transcripts 1, 2 and 3 are decreased in AD parietal cortex
  15. Discussion
  16. Acknowledgements
  17. References

Ten microliters of each RT-PCR reaction mixture was subjected to electrophoresis in a 0.018-g/mL agarose gel and analyzed by phosphorimagery using ImageQuant software (Molecular Dynamics, Sunnyvale, CA, USA). Quantitation was accomplished by placing rectangular cursors of fixed dimensions over each band and measuring pixel density for each sample, with local background subtraction. Four to five separate RT-PCR experiments were performed for each primer pair on each subject; the mean value of these experiments was used in the statistical analysis (Table 1). For transcripts with extremely low expression levels as indicated by pixel values, specifically transcripts 2, 4I, and 5U, only two RT-PCR experiments were averaged for each subject. For statistical analysis of group differences in transcript expression, a two-way anova was used [group (control versus AD subjects) × transcripts (1, 2, 3, 4, 4I, 4Ia, and 5U)], followed by two-tailed t-tests to determine significance between groups for each transcript. Box plot analysis identified extreme outliers in transcripts 2, 3 and 5U (Table 1). These outliers were eliminated from the two-way anova, post-hoc t-tests and Fig. 3. Comparisons of the effects of age and post-mortem interval on yield of total RNA were done by regression analysis and two-samples paired t-tests. All statistics were calculated and results graphed using Microsoft Excel (Microsoft, WA, USA) and SPSS 10.1 software (SPSS Inc., Chicago, IL, USA).

Table 1.  Results of RT-PCR experiments performed for each primer pair on each subject
 Transcript 1SEMTranscript 2SEMTranscript 3SEMTranscript 4SEMTranscript 4ISEMTranscript 4IaSEMTranscript 5USEM
  1. Values listed are pixel density values ± SEM. Numbers 1 through 24 in the vertical column identify the samples used with BDNF transcripts and standard error of the mean listed in the top horizontal row. The pixel values that are underlined are extreme outliers.

116 819 3343 033 611217 875130 14749 778 6628 502 2507 037 6621 047 331494 352110 7583 567 997820 650626 849246 297
225 821 9262 695 3491 735 690165 64253 196 9258 575 87610 474 5522 153 050566 53998 7535 736 379853 0752 687 693405 682
332 797 3012 500 2961 907 844173 99042 163 1587 777 09111 908 521812 804642 958165 0425 725 543386 0337 064 1461 121 427
428 543 946957 7081 754 551355 60442 009 5005 228 51111 301 671809 509259 67352325 503 1251 461 1832 314 29434 110
535 898 1232 147 628424 169116 07643 299 1256 898 19912 533 328846 855803 913462 0096 332 4811 153 024361 475257 062
632 187 7025 221 9642 060 237284 71539 695 1023 683 34510 208 729869 931338 96624 7195 870 8731 504 8721 002 167288 996
730 203 2035 233 968379 94638 62545 452 8814 157 8369 601 1822 302 385349 41342 1702 948 568512 800607 117305 044
824 701 2522 430 791937 371135 65127 730 76211 410 9589 272 5811 806 1701 991 1001 322 6434 034 720748 040507 186351 850
9548 283233 342003 085 447996 0421 354 477127 313439 410120 1393 331 9661 290 93023 59423 594
1020 750 8926 995 8865 414 304377 15733 633 9219 572 10712 496 8582 709 764340 300179 7155 324 6401 068 6093 607 137670 385
1162 36562 365778277823 745 050676 941975 521206 086217 47089 3762 478 577728 930145 50377 136
1210 655 8011 197 429264 11352 83729 318 2591 450 0614 154 594598 850450 621165 1163 801 774755 829723 875325 448
1317 162 1461 246 170147 600728440 373 2507 071 0016 962 680547 429360 101133 5264 860 465583 748553 968107 364
1423 458 5441 360 074399 089197 64142 397 2404 352 33910 673 1562 033 823398 68584 5716 576 002630 643971 938210 600
159 125 9261 290 66279 28317 13430 494 8064 613 8168 678 5791 109 594432 938153 5433 514 849622 728347 099162 303
166 115 309886 12716 400448112 872 2504 527 2585 255 0961 129 854498 59178 4943 152 6531 020 838551 649303 259
1728 013 521674 601807 37359 81129 970 8864 324 2659 351 7581 232 726624 85115 9285 789 8571 285 312377 79798 235
18826 278501 452103 96925262 892 247933 0192 520 9671 536 839548 652690 482 359 939996 3641 549 390346 536
1919 706 8461 486 934497 49069 60127 396 6532 341 1215 234 801576 683484 749165 2793 608 245994 0931 780 037348 233
2012 867 5011 435 986142 36846 54612 926 5832 557 5394 654 4492 158 951433 36099 8102 957 292239 7841 719 758505 184
215 994 249924 10455 282146421 411 677688 8092 224 165307 796629 057377 0122 461 246853 352552 120186 743
222 253 8842 098 65268 89047 08215 334 3322 734 2516 917 5913 147 661340 20141 3423 625 742135 023939 412746 414
236 357 706424 651253 15553 34721 239 5971 414 4232 238 072577 125453 42346 6992 328 355617 905827 129244 341
242 346 569405 80999 30552 0095 601 672890 3131 475 739334 767414 90562 5694 712 986637 767853 815357 443
image

Figure 3. Relative levels of mRNA for all brain-derived neurotrophic factor and β-actin transcripts in control versus Alzheimer disease samples. The y-axes show pixel intensity values determined by phosphorimage analysis. Error bars represent standard error of the mean. Statistically significant p-values are shown.

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Samples

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Human post-mortem brain tissue
  5. RNA isolation
  6. Primers for BDNF transcripts and β-actin
  7. RT-PCR
  8. Isolation and sequencing of RT-PCR products
  9. Quantitative and statistical analysis
  10. Results
  11. Samples
  12. New alternative splice site in transcript 4I
  13. β-Actin control
  14. Transcripts 1, 2 and 3 are decreased in AD parietal cortex
  15. Discussion
  16. Acknowledgements
  17. References

The samples consisted of 12 control and 12 AD post-mortem parietal cortex samples. The average age of the subjects in the control group was 77.58 ± 2.63 years and that of the AD group was 78 ± 2.49 years ( p = 0.9). The average post-mortem delay was 6.23 ± 0.039 h for the control group but only 3.05 ± 0.028 hours for the AD subjects ( p < 0.001). However, no significant differences were observed in the yield of total RNA extracted from both groups (485.62 ± 15.36 µg/g of tissue for control and 487.82 ± 29.23 µg/g of tissue for AD subjects, p = 0.93), or in the integrity of the purified RNA. Regression analysis yielded no significant correlation between yield of total RNA and age [r = 0.117 for control (p = 0.71) and r = 0.382 for AD (p = 0.22)]. Also, regression analysis for post-mortem delay and yield of total RNA resulted in no significant correlation [r = 0.312 (p = 0.35) for control and r= 0.037 (p = 0.91) for AD samples] (data not shown). We have previously demonstrated no significant correlation between BDNF mRNA content and age or post-mortem delay in both control and AD parietal cortex samples (Holsinger et al. 2000).

New alternative splice site in transcript 4I

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Human post-mortem brain tissue
  5. RNA isolation
  6. Primers for BDNF transcripts and β-actin
  7. RT-PCR
  8. Isolation and sequencing of RT-PCR products
  9. Quantitative and statistical analysis
  10. Results
  11. Samples
  12. New alternative splice site in transcript 4I
  13. β-Actin control
  14. Transcripts 1, 2 and 3 are decreased in AD parietal cortex
  15. Discussion
  16. Acknowledgements
  17. References

The reported size of transcript 4I is 414 bp (Aoyama et al. 2001). Upon PCR amplification, the 414 bp band was faintly present but was secondary in intensity to a 313-bp band (Fig. 1). Purification and sequence analysis of the 313-bp band revealed a new splice variant of exon 4I with splicing occurring 151 bp from the start of the 5′ primer and splicing out a 101-bp sequence (Fig. 2).

image

Figure 1. Ethidium bromide-stained gel showing RT-PCR products for transcript 4I. Lane 1 is the 100 bp DNA ladder. The faint band at 414 bp in lane 2 is the transcript 4I reported by Aoyama et al. (2001). The intense band at 313 bp in lane 2 is the newly discovered transcript 4Ia. The negative control, RT-PCR without reverse transcriptase, is in lane 3.

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image

Figure 2. Sequence of transcript 4Ia. The sequence shown is the human brain-derived neurotrophic factor transcript 4I (Aoyama et al. 2001). The underlined sequences at the ends indicate the primers used, and the middle underlined sequence is the 101 bp region that is spliced out of transcript 4I, resulting in the new transcript 4Ia. Note: The forward slash marks the start of the sequence of the mature coding exon 5.

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β-Actin control

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Human post-mortem brain tissue
  5. RNA isolation
  6. Primers for BDNF transcripts and β-actin
  7. RT-PCR
  8. Isolation and sequencing of RT-PCR products
  9. Quantitative and statistical analysis
  10. Results
  11. Samples
  12. New alternative splice site in transcript 4I
  13. β-Actin control
  14. Transcripts 1, 2 and 3 are decreased in AD parietal cortex
  15. Discussion
  16. Acknowledgements
  17. References

To control for variation between samples we used the constitutively expressed cytoskeletal protein, β-actin. Previous studies from ours and other laboratories have shown no significant difference in β-actin levels between normal and AD subjects (Takeda et al. 1991; Takeda et al. 1992; Holsinger et al. 2000). Our results support these previous findings; statistical comparisons between control and AD samples yielded no significant difference in β-actin mRNA levels (Fig. 3, p > 0.05).

Transcripts 1, 2 and 3 are decreased in AD parietal cortex

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Human post-mortem brain tissue
  5. RNA isolation
  6. Primers for BDNF transcripts and β-actin
  7. RT-PCR
  8. Isolation and sequencing of RT-PCR products
  9. Quantitative and statistical analysis
  10. Results
  11. Samples
  12. New alternative splice site in transcript 4I
  13. β-Actin control
  14. Transcripts 1, 2 and 3 are decreased in AD parietal cortex
  15. Discussion
  16. Acknowledgements
  17. References

The two-way anova revealed that overall expression of transcripts was significantly lower in AD patients compared with controls [for group, F6,162 = 4.998, p < 0.001)]. This is consistent with our previous results using the coding exon of BDNF as the RT-PCR target (Holsinger et al. 2000). Tests of between-subjects effects revealed a significant difference in individual transcripts. The difference was statistically significant for transcript 1 [F1,22 = 1.018, p = 0.027], transcript 2 [F1,20 = 31.674, p = 0.006] and transcript 3 [F1,20 = 2.115, p = 0.001], and at the border of statistical significance for transcript 4 [F1,22 = 1.255, p = 0.062]. None of the other transcripts [transcript 4I, F1,22 = 3.832, p = 0.456; transcript 4Ia, F1,22 = 0.126, p = 0.202; or transcript 5 U, F1,21 = 7.077, p = 0.547] demonstrated significant differences between control and AD (Fig. 3).

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Human post-mortem brain tissue
  5. RNA isolation
  6. Primers for BDNF transcripts and β-actin
  7. RT-PCR
  8. Isolation and sequencing of RT-PCR products
  9. Quantitative and statistical analysis
  10. Results
  11. Samples
  12. New alternative splice site in transcript 4I
  13. β-Actin control
  14. Transcripts 1, 2 and 3 are decreased in AD parietal cortex
  15. Discussion
  16. Acknowledgements
  17. References

Using RT-PCR on 24 age- and gender-matched control and AD samples from the parietal cortex, we report a significant decrease in three human BDNF mRNA transcripts in the parietal cortex of AD samples compared with controls. A two-way anova showed a significant effect for group and group × transcript. Post-hoc t-tests revealed a significant difference between control and AD samples for transcript 1,transcript 2 and transcript 3. None of the other transcripts (4, 4I, 4Ia or 5U) demonstrated any significant differences. We have previously shown that BDNF mRNA levels are decreased in the AD parietal cortex compared with controls (Holsinger et al. 2000). The decreased expression we demonstrate here in transcripts 1, 2 and 3 in AD could account for the decreased BDNF expression seen in previous studies examining the coding exon.

Six non-coding exons and their resulting transcripts have been reported for the human BDNF gene (exons 1, 2, 3, 4, 4I and 5U) (Aoyama et al. 2001). Within transcript 4I, we noted a new splice variant that was more highly expressed in parietal cortex tissue than the original 414 bp transcript 4I reported by Aoyama et al. (2001). Sequence analysis revealed a different splice variant of the 4I transcript (4Ia) containing a 101-bp deletion.

Although the regulatory elements and factors governing human BDNF expression are not known, we can draw parallels with the control of BDNF expression in the rat. In the rat, promoters I and III are both regulated by calcium (Tao et al. 1998; Tabuchi et al. 2000). Calcium influx leads to cAMP response element binding protein (CREB) phosphorylation, and phosphorylated CREB binds to and activates the cAMP response element (CRE) in rat BDNF promoter III (Shieh et al. 1998; Tao et al. 1998; Shieh and Ghosh 1999; West et al. 2001). Recently, promoter I was also reported to be CREB-dependent (Tabuchi et al. 2002). We have identified consensus CRE sites upstream of exons 1 and 3 in the human BDNF gene which suggests the human BDNF gene may be regulated in a manner similar to the rat gene. Levels of phosphorylated CREB are significantly decreased in post-mortem AD brain samples (Yamamoto et al. 1999), and a recent study demonstrates that Aβ(1–42) lowers CREB phosphorylation, causing decreased expression of the exon III BDNF transcript in rat cultured cortical neurons (Tong et al. 2001). Thus, our data implicating down-regulation of transcripts 1 and 3 in reduced BDNF expression in AD are consistent with known BDNF regulation in the rat. On the other hand, the regulatory factors and contribution to CNS BDNF expression for transcript 2 are still unknown.

In summary, we have shown here that only three of the seven human BDNF transcripts expressed in brain are down-regulated in AD. Whether the promoters governing these transcripts are regulated in a similar manner in the human CNS and the rat is still unknown. Further investigation will be necessary to identify the factors that regulate the human BDNF gene in AD.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Human post-mortem brain tissue
  5. RNA isolation
  6. Primers for BDNF transcripts and β-actin
  7. RT-PCR
  8. Isolation and sequencing of RT-PCR products
  9. Quantitative and statistical analysis
  10. Results
  11. Samples
  12. New alternative splice site in transcript 4I
  13. β-Actin control
  14. Transcripts 1, 2 and 3 are decreased in AD parietal cortex
  15. Discussion
  16. Acknowledgements
  17. References

This work was supported by grants from the Scottish Rite Charitable Foundation to DG and MF, and from the Ontario Neurotrauma Foundation to GY and MF. Also a special thanks goes to Dr Henry Szechtman, McMaster University, and Jennifer Dunn, University of Toronto, for their assistance with statistical analysis.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Human post-mortem brain tissue
  5. RNA isolation
  6. Primers for BDNF transcripts and β-actin
  7. RT-PCR
  8. Isolation and sequencing of RT-PCR products
  9. Quantitative and statistical analysis
  10. Results
  11. Samples
  12. New alternative splice site in transcript 4I
  13. β-Actin control
  14. Transcripts 1, 2 and 3 are decreased in AD parietal cortex
  15. Discussion
  16. Acknowledgements
  17. References
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