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A new brain-derived neurotrophic factor transcript and decrease
in brain-derived neurotrophic factor transcripts 1, 2 and 3 in
Alzheimer’s disease parietal cortex
Diego Garzon, Guanhua Yu and Margaret Fahnestock
Department of Psychiatry and Behavioural Neurosciences, McMaster University, Hamilton, Ontario, Canada
Abstract
Brain-derived neurotrophic factor (BDNF) supports hippo-
campal, 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 tran-
scripts. 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 phos-
phorylation is decreased in the Alzheimer’s disease brain.
This could lead to specific down-regulation of the brain-
derived neurotrophic factor transcripts shown here.
Keywords: brain-derived neurotrophic factor, gene expres-
sion, human, mRNA, RT-PCR.
J. Neurochem. (2002) 82, 1058–1064.
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 Alzhei-
mer’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 hippo-
campus 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, hippo-
campus 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
Received February 20, 2002; revised manuscript received May 10, 2002;
accepted May 13, 2002.
Address correspondence and reprint requests to Dr Margaret Fahne-
stock, Department of Psychiatry and Behavioural Neurosciences,
McMaster University, 1200 Main Street West, Hamilton, Ontario,
Canada L8N 3Z5. E-mail: [email protected]
Abbreviations used: AD, Alzheimer’s disease; BDNF, brain-derived
neurotrophic factor; CRE, cAMP response element; CREB, cAMP
response element binding protein.
Journal of Neurochemistry, 2002, 82, 1058–1064
1058 � 2002 International Society for Neurochemistry, Journal of Neurochemistry, 82, 1058–1064
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 givefour different transcripts. Downstream of the 3¢ exon thereare 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 exam-
ined 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.
Materials and methods
Human post-mortem brain tissue
Parietal cortex tissue samples from normal, neurologically
unimpaired subjects (n ¼ 12; six females, six males) and from
subjects with AD (n ¼ 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
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 exhib-
iting strong 28S and 18S ribosomal RNA bands on 0.01-g/mL
agarose gels were used for further analysis.
Primers for BDNF transcripts and b-actin
Human BDNF primer sequences were kindly provided by
Dr Mineyoshi Aoyama, Department of Bioregulation Research,
Nagoya City University Medical School, Mizuho-ku, Nagoya,
Japan. b-Actin primer sequences were previously described (StAmand et al. 1996). Primers were synthesized at the Central Facility
of the Institute for Molecular Biology and Biotechnology (MOBIX)
at McMaster University.
RT-PCR
For determination of transcripts 2 and 5U, RNA samples were
treated with DNaseI (1.0 lL/10 lg total RNA; Ambion, Austin, TX,USA) at 37�C for 30 min, followed by 2 lL of DNase Inactivationreagent. 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 lL aliquotsof the reverse transcriptase reaction mixture with 0.35 lM each ofthe 3¢ and 5¢ primers, 16.8 lCi of 33P-dCTP, and 2.5 U AmpliTaqGold (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 b-actin, 22 cycles. The amplification profile included aninitial 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 for7 min. For b-actin, annealing was at 64�C for 30 s and extension at72�C for 1 min. Four to five independent RT-PCRs were performedfor each primer pair.
Isolation and sequencing of RT-PCR products
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
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 Dynam-
ics, 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 signifi-
cance 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
Brain-derived neurotrophic factor transcripts 1059
� 2002 International Society for Neurochemistry, Journal of Neurochemistry, 82, 1058–1064
Tab
le1
Results
of
RT
-PC
Rexperim
ents
perf
orm
ed
for
each
prim
er
pair
on
each
subje
ct
Tra
nscript
1S
EM
Tra
nscript
2S
EM
Tra
nscript
3S
EM
Tra
nscript
4S
EM
Tra
nscript
4I
SE
MT
ranscript
4Ia
SE
MT
ranscript
5U
SE
M
116
819
334
3033
611
217
875
130
147
49
778
662
8502
250
7037
662
1047
331
494
352
110
758
3567
997
820
650
626
849
246
297
225
821
926
2695
349
1735
690
165
642
53
196
925
8575
876
10
474
552
2153
050
566
539
98
753
5736
379
853
075
2687
693
405
682
332
797
301
2500
296
1907
844
173
990
42
163
158
7777
091
11
908
521
812
804
642
958
165
042
5725
543
386
033
7064
146
1121
427
428
543
946
957
708
1754
551
355
604
42
009
500
5228
511
11
301
671
809
509
259
673
5232
5503
125
1461
183
2314
294
34
110
535
898
123
2147
628
424
169
116
076
43
299
125
6898
199
12
533
328
846
855
803
913
462
009
6332
481
1153
024
361
475
257
062
632
187
702
5221
964
2060
237
284
715
39
695
102
3683
345
10
208
729
869
931
338
966
24
719
5870
873
1504
872
1002
167
288
996
730
203
203
5233
968
379
946
38
625
45
452
881
4157
836
9601
182
2302
385
349
413
42
170
2948
568
512
800
607
117
305
044
824
701
252
2430
791
937
371
135
651
27
730
762
11
410
958
9272
581
1806
170
1991
100
1322
643
4034
720
748
040
507
186
351
850
9548
283
233
342
00
3085
447
996
042
1354
477
127
313
439
410
120
139
3331
966
1290
930
23
594
23
594
10
20
750
892
6995
886
5414
304
377
157
33
633
921
9572
107
12
496
858
2709
764
340
300
179
715
5324
640
1068
609
3607
137
670
385
11
62
365
62
365
7782
7782
3745
050
676
941
975
521
206
086
217
470
89
376
2478
577
728
930
145
503
77
136
12
10
655
801
1197
429
264
113
52
837
29
318
259
1450
061
4154
594
598
850
450
621
165
116
3801
774
755
829
723
875
325
448
13
17
162
146
1246
170
147
600
7284
40
373
250
7071
001
6962
680
547
429
360
101
133
526
4860
465
583
748
553
968
107
364
14
23
458
544
1360
074
399
089
197
641
42
397
240
4352
339
10
673
156
2033
823
398
685
84
571
6576
002
630
643
971
938
210
600
15
9125
926
1290
662
79
283
17
134
30
494
806
4613
816
8678
579
1109
594
432
938
153
543
3514
849
622
728
347
099
162
303
16
6115
309
886
127
16
400
4481
12
872
250
4527
258
5255
096
1129
854
498
591
78
494
3152
653
1020
838
551
649
303
259
17
28
013
521
674
601
807
373
59
811
29
970
886
4324
265
9351
758
1232
726
624
851
15
928
5789
857
1285
312
377
797
98
235
18
826
278
501
452
103
969
2526
2892
247
933
019
2520
967
1536
839
548
652
690
48
2359
939
996
364
1549
390
346
536
19
19
706
846
1486
934
497
490
69
601
27
396
653
2341
121
5234
801
576
683
484
749
165
279
3608
245
994
093
1780
037
348
233
20
12
867
501
1435
986
142
368
46
546
12
926
583
2557
539
4654
449
2158
951
433
360
99
810
2957
292
239
784
1719
758
505
184
21
5994
249
924
104
55
282
1464
21
411
677
688
809
2224
165
307
796
629
057
377
012
2461
246
853
352
552
120
186
743
22
2253
884
2098
652
68
890
47
082
15
334
332
2734
251
6917
591
3147
661
340
201
41
342
3625
742
135
023
939
412
746
414
23
6357
706
424
651
253
155
53
347
21
239
597
1414
423
2238
072
577
125
453
423
46
699
2328
355
617
905
827
129
244
341
24
2346
569
405
809
99
305
52
009
5601
672
890
313
1475
739
334
767
414
905
62
569
4712
986
637
767
853
815
357
443
Valu
es
liste
dare
pix
eld
ensity
valu
es
±S
EM
.N
um
bers
1th
rough
24
inth
evert
icalc
olu
mn
identif
yth
esam
ple
sused
with
BD
NF
transcripts
and
sta
ndard
err
or
ofth
em
ean
liste
din
the
top
horizonta
l
row
.T
he
pix
elvalu
es
that
are
underlin
ed
are
extr
em
eoutli
ers
.
1060 D. Garzon et al.
� 2002 International Society for Neurochemistry, Journal of Neurochemistry, 82, 1058–1064
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).
Results
Samples
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 ¼ 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 ± 15.36lg/gof tissue for control and 487.82 ± 29.23 lg/g of tissue for ADsubjects, p ¼ 0.93), or in the integrity of the purified RNA.
Regression analysis yielded no significant correlation between
yield of total RNA and age [r ¼ 0.117 for control (p ¼ 0.71)
and r ¼ 0.382 for AD (p ¼ 0.22)]. Also, regression analysis
for post-mortem delay and yield of total RNA resulted in no
significant correlation [r ¼ 0.312 (p ¼ 0.35) for control and
r ¼ 0.037 (p ¼ 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
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 splicingout a 101-bp sequence (Fig. 2).
b-Actin control
To control for variation between samples we used the
constitutively expressed cytoskeletal protein, b-actin. Previ-ous studies from ours and other laboratories have shown no
significant difference in b-actin levels between normal andAD 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 b-actin mRNAlevels (Fig. 3, p > 0.05).
Transcripts 1, 2 and 3 are decreased in AD parietal cortex
The two-way ANOVA revealed that overall expression of
transcripts was significantly lower in AD patients compared
Fig. 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.
Fig. 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.
Brain-derived neurotrophic factor transcripts 1061
� 2002 International Society for Neurochemistry, Journal of Neurochemistry, 82, 1058–1064
with controls [for group, F6,162 ¼ 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 signi-
ficant for transcript 1 [F1,22 ¼ 1.018, p ¼ 0.027], transcript 2
[F1,20 ¼ 31.674, p ¼ 0.006] and transcript 3 [F1,20 ¼ 2.115,
p ¼ 0.001], and at the border of statistical significance for
transcript 4 [F1,22 ¼ 1.255, p ¼ 0.062]. None of the other
transcripts [transcript 4I, F1,22 ¼ 3.832, p ¼ 0.456; transcript
4Ia, F1,22 ¼ 0.126, p ¼ 0.202; or transcript 5 U, F1,21 ¼7.077, p ¼ 0.547] demonstrated significant differences
between control and AD (Fig. 3).
Discussion
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
Fig. 3 Relative levels of mRNA for all brain-
derived neurotrophic factor and b-actin
transcripts in control versus Alzheimer
disease samples. The y-axes show pixel
intensity values determined by phosphorim-
age analysis. Error bars represent standard
error of the mean. Statistically significant
p-values are shown.
1062 D. Garzon et al.
� 2002 International Society for Neurochemistry, Journal of Neurochemistry, 82, 1058–1064
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) phos-
phorylation, and phosphorylated CREB binds to and acti-
vates 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 Ab(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
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.
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