Download Supplementary Information (doc 137K)

Yiu et al.,
Supplemental Data
Increasing CREB function in the CA1 region of dorsal hippocampus
rescues the spatial memory deficits in a mouse model of Alzheimer’s disease
Adelaide P. Yiu, HBSc, Asim J. Rashid, PhD, Sheena A. Josselyn, PhD
Supplemental Figures S1-S8
1
Yiu et al.,
Supplemental Data
SUPPLEMENTAL DATA
Figure S1. The disruption of CREB activation observed in the CA1 region of dorsal
hippocampus of Tg mice was specific in terms of brain region and CREB activation
In Figures 1d and 1e, we showed that Tg mice had fewer pCREB+ neurons in the
CA1 region of dorsal hippocampus than WT littermate mice across the three behavioral
treatments (homecage, novel context exposure and watermaze). To examine the
anatomical specificity of this deficit, we also examined pCREB levels in the DG in these
mice. Because DG cells are tightly packed, we used stereological counting methods to
assess the number of pCREB+ neurons. We observed no difference in pCREB levels in
the DG between Tg and WT mice following any behavioral treatment (Figure S1a, b).
This finding was confirmed by the results of a Genotype (Tg, WT) X Treatment
(homecage, novel context, watermaze) ANOVA that revealed no significant effect of
Treatment (F2,18 = 0.74; p > .05), Genotype (F1,18 = 0.003; p > .05) or Genotype X
Treatment interaction (F2,18 = 0.55; p > .05). Therefore, Tg mice show normal CREB
function in the DG. This finding contrasts the deficit in pCREB levels observed in the
CA1 region.
To further examine the deficit in CREB activation in the CA1 region of dorsal
hippocampus of Tg mice we also examined overall levels of CREB (both phosphorylated
and non-phosphorylated). We used an antibody that recognizes both phosphorylated and
unphosphorylated CREB (tCREB) and examined Tg and WT littermate mice following
exposure to a novel context. We observed no difference in density of tCREB+ neurons in
the CA1 region of dorsal hippocampus in Tg and WT mice (Figure S1c, d; F1,14 = 0.51; p
2
Yiu et al.,
Supplemental Data
> .05). This finding agrees with our results from a Western blot analysis which showed
that although pCREB levels were decreased in the dorsal hippocampus of Tg mice, total
levels of CREB protein were normal (Figures 1g, 1h). Together, these results show that
Tg mice have a specific disruption of CREB activation in the CA1 region of dorsal
hippocampus.
Figure S2. Increasing CREB function in the CA1 region of dorsal hippocampus in
Tg mice during watermaze training; additional watermaze measures
Figures 2b and 2c show that Tg mice have spatial memory deficits (assessed by
more time spent in the target versus other zones in a probe test) that are rescued by
microinjecting CREB vector in the CA1 region of the dorsal hippocampus. We also
measured a number of additional variables over the course of training [including latency
to reach the platform, swim speed, distance traveled and thigmotaxis (swimming in the
perimeter of the pool)] and testing (swim speed, thigmotaxis, time spent in the 4 zones of
the pool and 4 quadrants of the pool) (Figure S2).
Training data
Time to platform
Over the 3 days of training, the latency to reach the platform declined in all
groups, but the latency was higher overall in Tg mice microinjected with GFP vector
(Figure S2a). The results of a Group (WT-GFP, WT-CREB, Tg-GFP, Tg-CREB) X Day
(3) ANOVA revealed significant main effects of Group (F3,51 = 2.93; p < .05) and Day
(F2,102 = 90.43; p < .001) and no significant Group X Day interaction (F6,102 = 1.74; p
3
Yiu et al.,
Supplemental Data
> .05). Post-hoc Newman-Keuls tests confirmed that over days, all groups showed a
decrease in latency to reach the platform, but that the Tg-GFP group have longer
latencies than the WT-GFP mice (p = .055). Therefore, over training, all groups except
the Tg group with GFP vector, showed shorter latencies, consistent with the adoption of a
more focal search strategy and the formation of spatial memory.
Swim speed
Over training, there was a tendency for groups to swim faster (Figure S2b). The
results of Group X Day ANOVA confirm this interpretation, showing a significant effect
of Day (F2,102 = 4.64; p < .05) and Group X Day interaction (F6,102 = 2.32; p < .05) but no
significant effect of Group (F3,51 = 1.05; p > .05). Post-hoc analysis showed a trend for
all mice to swim faster on the second and third days of training.
Distance travelled
As can be seen in Figure S2c, all groups showed a decrease in the distance
travelled over training days but this decrease was attenuated in Tg mice with GFP vector.
A Group X Day ANOVA revealed significant effects of Group (F3,51 = 3.01; p < .05) and
Day (F2,102 = 41.90; p < .001) but no significant Group X Day interaction (F6,102 = 1.35; p
> .05). Post-hoc tests showed that Tg mice with GFP vector tended to travel greater
distances.
Thigmotaxis
4
Yiu et al.,
Supplemental Data
Finally, we examined the levels of thigmotaxis (swimming in the perimeter of the
pool) over training. We observed that over training days, thigmotaxis decreased in all
groups (Figure S2d). An ANOVA showed significant effects of Day (F6,102 = 150.50; p
< .001) only, and no significant Group X Day interaction (F6,102 = 1.06; p > .05) or main
effect of Group (F3,51 = 1.15; p > .05). Therefore, all groups showed decreased
thigmotaxis over training.
Probe data
The decrease in time to reach the platform and distance travelled over training
days by all groups except for the Tg-GFP group may reflect the adoption of a focal search
strategy (in that mice search swim towards the platform with little variance) or the
adoption of other non-spatial strategies (Gallagher et al., 1993; Gass et al., 1998; Lipp &
Wolfer, 1998; Clapcote & Roder, 2004). To discriminate between a spatial and nonspatial strategy and to specifically examine the formation of spatial memory, we gave
mice a probe test at the end of training in which the platform was removed from the pool.
The results of a zone analysis (target versus the average of the three other zones)
conducted on the probe test are shown in Figure 2b. Additionally, we examined a
number of other measures from the probe test.
Swim speed and thigmotaxis
There was no difference in swim speed during the probe test between the groups
(Figure S2e; one-way ANOVA; F3,51 = 2.73; p > .05). Similarly, the levels of thigmotaxis
during the probe did not differ between groups (Figure S2f; F3,51 = 0.09; p > .05).
5
Yiu et al.,
Supplemental Data
Time spent in each of the four zones
We also analyzed the probe data by examining the time mice spent in each of the
four zones, rather than comparing the time spent in the target zone versus the average of
the three other zones (as in Figure 2b). During this probe test, WT-GFP, WT-CREB and
Tg-CREB mice spent more time in the zone in which the platform was located during
training [target zone (T)] than in the other equally-sized areas of the pool (zones 1,2,3),
while Tg-GFP mice failed to selectively search in the target zone (Figure S2g). A Group
X Zone (T,1,2,3) ANOVA revealed a significant interaction between Group and Zone
(F9,153 = 3.59; p < .001), as well as significant main effects of Zone (F3,153 = 51.49; p
< .001) and Group (F3,51 = 7.05; p < .001) alone. Post hoc comparisons performed on the
significant interaction showed that while WT-CREB, WT-GFP and Tg-CREB mice
searched selectively in the target zone compared to each of the other 3 zones, Tg-GFP
mice showed no such preference (although these mice did spend more time in the T zone
versus zone 1). Therefore, using this additional measure of probe test performance, TgGFP mice show profound deficits in the formation of a spatial memory that, importantly,
were reversed by CREB vector.
Time spent in each of four quadrants
We similarly examined probe test data looking at the percent time mice spent in
each quadrant of the pool. Consistent with the zone analysis, all groups (except Tg-GFP
mice) spent more time in the target quadrant than in the other 3 quadrants of the pool
[Opposite (O), left (L), right (R)] (Figure S2h). A Group X Quadrant (4) ANOVA
6
Yiu et al.,
Supplemental Data
revealed a significant interaction (F9,153 = 4.89; p < .001), as well as significant main
effects of Quadrant (F3,153 = 29.76; p < .001), but not Group (F3,51 = 0.27; p >.05). Post
hoc comparisons showed that while WT-CREB, WT-GFP and Tg-CREB mice searched
selectively in the target quadrant, Tg-GFP mice showed no such preference and spent
equal time in each quadrant. This interpretation was confirmed by an ANOVA
specifically comparing time spent in the target quadrant between Groups (F3,51 = 10.24; p
< .001). Therefore, using an additional measure of spatial memory formation in the
watermaze, Tg mice show profound deficits that are rescued by increasing CREB levels.
Figure S3. Increasing CREB function in the DG of Tg mice; additional watermaze
measures
We observed that the spatial memory deficits in Tg mice were rescued by
microinjecting CREB vector into the CA1 region of the dorsal hippocampus. To examine
the anatomical specificity of this rescue, we compared the performance of mice
microinjected with CREB vector into the CA1 region to mice similarly microinjected
with CREB vector into the DG.
Training data
Time to platform
Over training, the time to reach the platform decreased in all groups [(WT-DGCREB (n=5), WT-CA1-CREB (from above), Tg-DG-CREB (n=10), Tg-CA1-CREB
(from above)] but this tendency was attenuated in Tg mice with CREB vector
microinjected into the DG (Figure S3a). The results of a Group X Day ANOVA showed
7
Yiu et al.,
Supplemental Data
no significant Group X Day interaction (F6,72 = 1.91; p > .05), but significant effects of
Day (F2,72 = 64.58; p < .001) and Group (F3,36 = 3.11; p < .05).
Swim speed
Swimming speed tended to increase over training days in all groups (Figure S3b;
significant main effect of Day (F2,72 = 6.23; p < .01) and no significant effect of Group
(F3,36 = 0.611; p > .05) or interaction (F6,72 = 1.63; p > .05)]. Newman-Keuls tests
indicated that, in general, all mice showed faster swim speed over training days.
Distance travelled
The distance travelled decreased over training days in all groups but this was
diminished in Tg-DG-CREB mice [Figure S3c; significant effect of Day (F2,72 = 34.24; p
< .001) and Group (F3,36 = 5.14; p < .05) but no significant Group X Day interaction
(F6,72 = 1.45; p > .05)]. Post-hoc tests showed that overall mice decreased the distances
travelled over days but that Tg-DG-CREB mice travelled farther than WT-DG-CREB
and WT-CA1-CREB mice.
Thigmotaxis
Over training, thigmotaxis decreased in all groups (Figure S3d). The results of
statistical analysis confirms this interpretation; an ANOVA showed no significant Group
X Day interaction (F6,72 = 1.10; p > .05), or main effect of Group (F3,36 = 1.10; p > .05)
but a significant effect of Day (F6,72 = 61.03; p < .001).
8
Yiu et al.,
Supplemental Data
Probe data
Swim speed and thigmotaxis
There was no difference in swim speed between the groups during the probe test
(Figure S3e; F3,36 = 0.92; p > .05). Similarly, the levels of thigmotaxis during the probe
did not differ between groups (Figure S3f; F3,36 = 0.04; p > .05).
Time spent in each of the four zones
Figure S3g shows that time mice spent in each of the four zones during the probe
test. As can be observed from this graph, all groups spent more time in the target zone
than in the three other equally-sized zones except for Tg-DG-CREB mice, which failed to
selectively search in the target zone. A Group X Zone ANOVA revealed a significant
interaction (F9,108 = 6.12; p < .001), as well as main effect of Zone (F3,108 = 38; p < .001),
but not Group (F3,36 = 2.44; p > .05). Post hoc comparisons revealed that while WT-DGCREB, WT-CA1-CREB and Tg-CA1-CREB mice searched selectively in the target zone,
Tg-DG-CREB mice showed no such preference. Therefore, increasing CREB function in
the DG did not rescue the profound deficits in the formation of a spatial memory in Tg
mice.
Time spent in each of the four quadrants
Consistent with the zone analysis, all groups (except the Tg-DG-CREB group)
spent more time in the target quadrant of the pool than in the other 3 quadrants (Figure
S3h). A Group X Quadrant ANOVA revealed a significant interaction (F9,108 = 5.93; p
< .001), as well as a significant main effects of Quadrant (F3,108 = 26.57; p < .001) but no
9
Yiu et al.,
Supplemental Data
significant effect of Group (F3,36 = 5.97; p > .05) alone. Post hoc comparisons showed
that while WT-DG-CREB, WT-CA1-CREB and Tg-CA1-CREB mice searched
selectively in the target quadrant, Tg-DG-CREB mice showed no such preference. This
interpretation was confirmed by an ANOVA specifically comparing the time spent in the
target quadrant between Groups (F3,36 = 11.79; p < .001). In agreement with other
measures, therefore, Tg-DG-CREB mice show profound deficits in the formation of a
spatial memory.
Figure S4. Microinjecting CREB vector restored CREB activation in the CA1
region of dorsal hippocampus in Tg mice
Tg mice have disrupted CREB activity in the CA1 region of dorsal hippocampus
(see Figure 1e). To examine whether microinjecting CREB vector into this region
restored CREB activity, we examined pCREB levels in Tg mice microinjected with
CREB vector. We observed that the disruption in CREB activity in Tg mice after the
watermaze probe test was reversed by microinjecting CREB vector into the CA1 region
(Figure S4). An ANOVA with between factor Group (Tg-GFP, Tg-CREB, WT-GFP,
WT-CREB) revealed a significant effect (F3,28 = 6.65; p < .001), and post-hoc analysis
showed that only Tg mice microinjected with GFP vector (Tg-GFP mice) had
significantly lower pCREB levels than WT mice microinjected with control vector (WTGFP). Importantly, pCREB levels in Tg-CREB mice did not differ from WT mice.
Therefore, microinjecting CREB vector into dorsal hippocampus restored CREB
activation in Tg mice.
10
Yiu et al.,
Supplemental Data
Figure S5. Increasing CREB function in the CA1 region of dorsal hippocampus in
CaMKII∆-/- mice; additional watermaze measures
Training data
Time to platform
Over training, the time to reach the platform decreased in WT mice (regardless of
vector) but did failed to decrease in CaMKII∆-/- mice (regardless of vector) (Figure S5a).
The results of a Group (WT-GFP, WT-CREB, CaMKII∆-/- -GFP, CaMKII∆-/- -CREB)
X Day ANOVA confirmed this interpretation, showing a significant Group X Day
interaction (F6,64 = 3.05; p < .05), as well as a significant effect of Day (F2,64 = 11.69; p
< .001) and Group (F3,32 = 3.81; p < .05).
Swim speed
Swim speed was similar over training, but WT-CREB mice tended to swim faster
over days (Figure S5b). The results of a Group X Day ANOVA showed a significant
interaction (F6,64 = 2.36; p < .05), but no significant effect of Group (F3,32 = 2.01; p > .05)
or Day (F2,72 = 1.78; p > .05). Post-hoc Newman-Keuls tests on the significant interaction
show that WT-CREB mice swim faster on training day 3 compared to day 1.
Distance travelled
The distance travelled tended to decrease over training days, although this effect
was not as pronounced in CaMKII∆-/--CREB mice (Figure S5c). Statistical analysis
revealed no significant Group X Day interaction (F6,64 = 0.68; p > .05), but significant
11
Yiu et al.,
Supplemental Data
main effects of Day (F2,64 = 6.19; p < .05) and Group (F3,32= 3.21; p < .05). Post-hoc
tests showed that all groups travelled less over days and that CaMKII∆-/- CREB mice
tended to travel longer distances compared to WT mice.
Thigmotaxis
The levels of thigmotaxis decreased over training days in WT mice (regardless of
vector) but not in CaMKII∆-/- mice (with either GFP or CREB vector) (Figure S5d).
The ANOVA showed significant main effect of Group (F3,32 = 5.77; p < .05) and Day
(F2,64 = 11.94; p < .001) but no significant interaction (F6,64 = 1.28; p > .05).
Probe data
Swim speed and thigmotaxis
There was no difference in swim speed between the groups during the probe test
(Figure S5e; F3,32 = 2.55; p > .05). However, the levels of thigmotaxis during the probe
differed between groups (Figure S5f; F3,32 = 25.67; p < .001). CaMKII∆-/- mice
(regardless of vector) had significantly higher levels of thigmotaxis compared to WT
mice (regardless of vector).
Time spent in each of the four zones
WT mice (regardless of vector) spent more time in the target zone than in the
other three zones while CaMKII∆-/- mice (regardless of vector) showed equal preference
for the four zones (Figure S5g). A Group X Zone ANOVA revealed a significant
interaction (F9,96 = 3.21; p < .001), as well as significant main effects of Zone (F3,96 =
12
Yiu et al.,
Supplemental Data
11.41; p < .001) and Group (F3,32 = 7.79; p < .001). Post hoc comparisons confirmed that
while WT-GFP and WT-CREB mice searched selectively in the target zone, CaMKII∆-GFP and CaMKII∆-/- -CREB mice showed no such preference. These data confirm
/-
those presented in Figure 3d.
Time spent in each of the four quadrants
A similar pattern emerged when we examined the time mice spent in each of the
four quadrants of the pool. WT mice spent more time in target quadrant than in the other
quadrants, while CaMKII∆-/- mice failed to selectively search in the target quadrant
(Figures S5h). A Group X Quadrant ANOVA revealed a significant interaction (F9,96 =
2.97; p < .001), but no significant main effects of Quadrant (F3,96 = 1.65; p > .05) or
Group (F3,32 = 0.52; p > .05) alone. Post hoc comparisons showed that while WT-GFP
and WT-CREB mice searched selectively in the target quadrant, CaMKII∆-/--GFP and
CaMKII∆-/--CREB mice showed no such preference. This interpretation was confirmed
by an ANOVA specifically comparing time spent in the target quadrant between Group
(F3,32 = 6.37; p < .001). Therefore, unlike Tg mice, increasing CREB in the CA1 of
CaMKII∆-/- mice did not rescue their profound deficits in the formation of a spatial
memory.
Figure S6. Microinjecting CREB vector restored spatial memory in Tg mice without
affecting A plaque load or levels
In Figure 2b we showed that microinjecting CREB vector into the CA1 region of
dorsal hippocampus rescued the spatial memory deficits in Tg mice. To address the
13
Yiu et al.,
Supplemental Data
potential mechanisms underlying this rescue, we first examined different measure of A
in these mice. There are many forms of Aβ: monomers, multimers (varying from dimers
to dodecamers that are collectively called oligomers) and protofibrillar forms in which
Aβ is aggregated in a beta-pleated sheet structure, and finally into mature fibrils, which
form amyloid plaques (Selkoe, 1994; Jan et al., 2010).
First, we assessed whether CREB vector decreased plaque load in Tg mice. As
expected we observed no plaques in WT mice. In addition, we found that the percent
volume occupied by plaques in either the dorsal hippocampus (Figure S6a, F2,25 = 0.095;
p > .05) or frontal cortex (Figure S6b, F2,25 = 0.01; p > .05) of Tg mice was unaffected by
microinjecting CREB vector [ANOVAs compared Tg-no surgery (n=17), Tg-GFP (n=5)
and Tg-CREB (n=6) mice].
We next asked whether microinjecting CREB vector decreased the high levels of
aggregated Ain Tg mice. Again, WT control mice showed low levels of aggregated
AFigure S6c). Moreover, CREB vector did not decrease the high levels of aggregated
A observed in Tg mice [ANOVA comparing Tg-no surgery (n=2), Tg-GFP (n=1) and
Tg-CREB (n=3) showed no difference; F3,3 = 1.94; p > .05]. A similar analysis on the
levels of soluble in the dorsal hippocampus revealed that the high levels observed in
Tg mice were also unaffected by CREB vector Figure S3d, F3,3 = 0.71; p
> .05).Therefore, increasing CREB function in the CA1 region of dorsal hippocampus
did not rescue the memory deficits in Tg mice by decreasing Aplaque load, aggregated
or soluble forms of A. The finding that Tg mice can show normal spatial memory yet
high plaque load is in keeping with reports that humans can show intact cognitive
14
Yiu et al.,
Supplemental Data
function despite having high Aβ plaque loads (that meet or exceed the criteria for AD
diagnosis) (Katzman, 1988).
Figure S7. Levels of viral infection
We performed several analyses to examine the level of infection produced by
microinjection of CREB or GFP vector into WT or Tg mice (Figure S7). Examples of a
robust and sparse infection, as indicated by GFP fluorescence, are presented in Figure
S7a. We observed that the total infected area did not differ across vector or mouse
genotype (Figure S7b; F3,8 = 1.29; p > .05; WT-GFP, WT-CREB, Tg-GFP, Tg-CREB)
nor did the percentage of the dorsal hippocampal CA1 region that was infected (Figure
S7c; F3,8 = 0.13; p > .05). Only those mice that showed robust bilateral infection of the
CA1 region of dorsal hippocampus (< 20% of CA1 region infected) were included in
subsequent statistical analyses. We confirmed these results using stereological counting
methods. The estimated number of neurons infected (Fig. S7d; F3,8 = 0.26; p > .05) or the
percent CA1 neurons infected (Figure S7e; F3,8 = 0.93; p > .05) did not differ across
vector or group.
Figure S8. Verifying p1005 vectors
To analyze dendritic complexity and synapse density in Figure 5, we constructed
two viral vectors (Figure S8a). This was critical because in our original CREB vector,
GFP was fused to CREB and therefore largely localized to the nucleus. Therefore we
constructed a new vector in which CREB and GFP were bi-cistronically expressed from
two different promoters. Specifically, in the p1005-CREB amplicon CREB expression is
15
Yiu et al.,
Supplemental Data
driven by viral IE4/5 and GFP expression is driven by the CMV promoter. In this way,
GFP fills the cell, allowing us to examine dendritic morphology. As a control we use the
p1005 amplicon-based vector in which GFP expression is driven by the CMV promoter.
Because the CMV promoter contains several CRE-sites to which CREB may bind, we
considered the possibility that there could be increased GFP expression from the p1005CREB amplicon compared to the p1005 amplicon. To test whether higher levels of GFP
were produced by the p1005-CREB vector, we infected HEK 293 cells in culture with
equal amounts of p1005-CREB or p1005 vector. We observed similar GFP fluorescence
in p1005 and p1005-CREB treated cells (Figure S8b, both in number of GFP-expressing
cells and average intensity of fluorescence in each cell). Western blotting of cell lysates
confirmed this. Although levels of CREB expression were higher in cells infected with
p1005-CREB virus than p1005 virus, levels of GFP were not (Figure S8c). Therefore,
GFP expression from each amplicon was equivalent and not affected by the presence of
exogenous CREB expressed from the viral vector.
SUPPLEMENTAL EXPERIMENTAL METHODS
pCREB levels in the DG
To examine pCREB levels (density of pCREB+ neurons) in the DG, we
performed stereological counting using Stereology Investigator software (MBF) on at
least 6 sections per mouse from a subset of the mice in each behavioral treatment group
(homecage Tg n=3, WT n=3; novel context exposure Tg n= 4, WT n=4; and watermaze
Tg n= 4, WT n=4; Figure S1a). The DG was traced and the optical fractionator probe was
16
Yiu et al.,
Supplemental Data
used to randomly place sampling boxes (250 m x 250 m) throughout the DG (West et
al., 1991) and the number of pCREB+ cells per section were counted.
tCREB levels in the CA1 region of dorsal hippocampus
To assess the overall levels of total CREB (both phosphorylated and
unphosphorylated; tCREB) we examined alternate sections from mice in the novel
context exposure group (Tg n= 7, WT n = 7; Figure S1b). Sections were treated similarly
except we used a primary antibody (mouse anti-CREB, 1:2,000; Chemicon International,
Temecula, CA MAB5432) that recognizes total CREB and a secondary antibody (715066-151, biotin-anti-mouse IgG, Dnky, 1:500; Jackson ImmunoResearch, West Grove,
PA). The number of tCREB+ nuclei was assessed by two experimenters unaware of the
treatment condition using Image J software and a density score was calculated as above.
pCREB levels following CREB vector microinjection into the CA1 region of dorsal
hippocampus in Tg mice
To examine whether CREB vector normalized CREB activity in Tg mice (Figure
S2), we microinjected CREB or GFP vector into the CA1 region of dorsal hippocampus
of Tg and WT mice and trained them in the watermaze. Following the probe test, we
assessed the density of pCREB+ cells in the CA1 region, as before (n=8 for all groups).
Aggregated and monomeric Alevels
To examine whether increasing CREB function reduced levels of oligomeric and
fibrillar aggregates of Agenerally considered to be the toxic forms of Aand major
17
Yiu et al.,
Supplemental Data
contributors to brain dysfunction in AD), we used a sample enrichment protocol
(Amorfix Aggregated A Assay, A4) that detects aggregated A at time points before
immunohistochemical techniques are able to detect plaques. Dorsal hippocampal tissue
from Tg mice microinjected with CREB (n=3) or GFP (n=1) vectors as well as Tg (n=2)
and WT (n=2) mice without surgery was isolated and homogenized in cell lysis buffer
(50 mM Tris, 0.25 M sucrose, 25 mM KCl, 5 mM MgCl2with protease and phosphatase
inhibitors). A oligomers (rather than monomers) were then specifically isolated and
disaggregated to allow detection of (now) monomeric A(Tanghe et al., 2010).
Briefly,A was detected by immunoassay using europium-fluorescent beads coupled to
the mouse monoclonal 4G10 antibody (N-terminal, aa 1-17) and magnetic beads coupled
to the antibodies 1F8 (C-terminal, aa 30-40 of A peptide) and 2H12 (C-terminal, aa 3042) which recognizes human A0 and A42, respectively. The intensity of the europium
fluorescent signal was measured using time resolved fluorescence (TRF) on each sample
in triplicate and was taken as being directly proportional to the concentration of
aggregated A within the sample. The limit of detection using this technique is 50 fg of
protein per well. The values presented on the graph are europium counts from a 1:2,000
dilution of homogenate.
Monomeric A was assessed using the above samples. Briefly, samples were
diluted in PBS-TB in order to provide a signal within the linear range of the assay.
Diluted samples were incubated with europium-fluorescent beads coupled to 4G10
antibody and magnetic beads coupled to antibodies 1F8 and 2H12 (as above) at 37oC
with shaking. Following incubation, samples were placed on a magnet to isolate the
immune complex. The europium fluorescence intensity was measured using TRF on each
18
Yiu et al.,
Supplemental Data
sample in triplicate, and is directly proportional to the concentration of A within the
sample. The limit of detection was 50 fg/well. The S/N cut-off value for all experiments
was 2.0, equaling two times the background signal from buffer alone.
Estimating number of neurons infected by viral vectors
To examine the number of CA1 pyramidal neurons in the dorsal hippocampus
infected by CREB and GFP vectors, we performed stereological counting using
Stereology Investigator software on at least 6 sections per mouse from a subset of mice
(Tg-GFP n=3, Tg-CREB n=3, WT-GFP n=3, WT-CREB n=3). The CA1 region of the
dorsal hippocampus was traced and the optical fractionator probe used to randomly place
sampling boxes (250 m x 250 m) throughout the CA1 (West et al., 1991) and the
number of GFP+ cells were assessed. The area of the traced CA1 infected region was also
calculated using the optical fractionator probe. This process was repeated to assess the
number of nuclei in the CA1 and the area. The percent GFP+ cell density was calculated
by dividing GFP+ cell density by the DAPI+ cell density. Similarly, the percent area of
CA1 infected was calculated by dividing the CA1 GFP+ infected area by the total CA1
region of the dorsal hippocampus traced area.
Equivalent levels of GFP expression from p1005 and p1005-CREB amplicons
HEK 293 cells were seeded in 60 mm2 dishes and used for virus infection at 80%
confluency. HSV amplicons p1005 or p1005-CREB were diluted in 3 ml DMEM
(InVitrogen) to a concentration of 1 x 107 infectious units/ml and added to cells.
Following incubation for 3 hr at 370C, the virus-containing media was removed and
19
Yiu et al.,
Supplemental Data
replaced with normal growth media (DMEM, 5% FBS, 20 mM glutamine). After
incubation for another 17 hrs at 370C, cells were harvested and briefly sonicated in 200 l
buffer containing 50 mM Tris.Cl pH7.5, 0.25 M sucrose, 25 mM KCl, 5 mM MgCl2 and
protease inhibitors. Protein lysates were further processed and subjected to Western
blotting as described in Materials and Methods. After initial blotting with antibodies
against CREB and GFP, PVDF membranes were stripped by incubation in 0.2 M glycine,
0.05% Tween-20, pH 2.5, for 1 hr at 500C and then incubated with an antibody against
GAPDH. Primary antibodies used were as follows: mouse anti-CREB, 1:1000 (Chemicon,
Temulca, CA); rabbit anti-GFP, 1:1000 (InVitrogen); rabbit anti-GAPDH, 1:5000 (Cell
Signaling Inc.). Virus titers were previously determined through serial dilution and
infection of 293 cells followed by counting of fluorescent cells.
20
Yiu et al.,
Supplemental Data
SUPPLEMENTAL FIGURE LEGENDS
Figure S1. Tg mice show a specific deficit in CREB activation in the CA1 region of
dorsal hippocampus
(a) Representative photomicrographs showing that Tg mice have normal CREB
activation (pCREB+ nuclei) in the DG following novel context exposure. Images are
quantified in (b).
(b) Number of pCREB+ nuclei in the DG (estimated by stereological counting) is similar
in Tg and WT mice across three different behavioral treatments [homecage (HC), novel
context exposure (CX), or training and testing in the watermaze (WM)].
(c) Representative photomicrographs showing that Tg mice have normal total CREB
levels (tCREB+ nuclei) in the CA1 region of the dorsal hippocampus following novel
context exposure. Images are quantified in (d).
Figure S2. Increasing CREB function in the CA1 of dorsal hippocampus of Tg mice
during watermaze training; additional measures
In Figures 2b,c Tg or WT littermate mice were microinjected GFP or CREB vector into
the CA1 region of the dorsal hippocampus, trained and then tested in the watermaze
[WT-GFP (n=16), WT-CREB (n=14), Tg-GFP (n=14), Tg-CREB (n=11) mice]. All data
are mean ± SEM.
(a-d) Measures of performance over training.
(a) Time to platform. All groups, except Tg-GFP, showed decreased latency over
training.
21
Yiu et al.,
Supplemental Data
(b) Swim speed. Tg mice did not show a difference in swim speed, compared to WT
mice. Groups showed similar swim speed and all groups tended to swim faster on the
second and third day of training.
(c) Distance travelled. Groups showered shorter distance travelled across training days,
but this effect was attenuated in Tg-GFP mice.
(d) Thigmotaxis. The tendency to swim near the pool perimeter decreased in all groups
across training days.
(e-h) Measures of performance during the probe test.
(e, f) Swim speed and thigmotaxis were similar in all groups during the probe test
(g) Examining the probe trial using 4 separate zones. Similar to the analysis examining
target versus the average of the 3 other zones, all groups except Tg mice with GFP vector
spent more time in the zone of the pool where the platform was previously located (target
zone, T) than in the other 3 equally sized zones (zone 1,2,3).
(h) Examining the probe trial using 4 quadrants. Similarly, all groups except Tg-GFP
spent more time in the quadrant of the pool where the platform was previously located
than in the other quadrants [Opposite (O), Left (L), Right (R)] of the pool. Therefore, Tg
mice have deficits in the formation of spatial memory, assessed by several variables, and
this deficit is reversed by microinjecting CREB vector.
Figure S3. Increasing CREB function in the DG of Tg mice during watermaze
training; additional measures
22
Yiu et al.,
Supplemental Data
In Figures 3b,c Tg or WT littermate mice were microinjected CREB vector into the DG,
trained and then tested in the watermaze [results are compared to mice that were
microinjected with CREB into the CA1 region, above)].
(a-d) Measures of performance over training.
(a) Time to platform. All groups showed decreased latency to reach the platform over
training but this was attenuated in Tg-DG-CREB mice.
b) Swim speed. Groups showed similar swim speed and all groups tended to swim faster
over training.
(c) Distance travelled. Groups showered shorter distance travelled across training days,
but this effect was attenuated in Tg-DG-CREB mice.
d) Thigmotaxis. All groups showed decreasing levels of thigmotaxis over training days.
(e-h) Measures of performance during the probe test.
(e, f) Swim speed and thigmotaxis were similar in all groups during the probe test
(g) Examining the probe trial using 4 separate zones. Similar to the analysis examining
target versus the average of the 3 other zones, all groups except Tg mice with CREB
vector microinjected into the DG spent more time in the zone of the pool where the
platform was previously located (target zone, T) than in the other 3 equally sized zones
(zone 1,2,3).
(h) Examining the probe trial using 4 quadrants. Similarly, all groups except the Tg-DGCREB group spent more time in the quadrant of the pool where the platform was
previously located than in the other quadrants [Opposite (O), Left (L), Right (R)] of the
pool. Therefore, the rescue of the spatial memory deficit in Tg mice by CREB vector
shows anatomical specificity.
23
Yiu et al.,
Supplemental Data
Figure S4. Microinjecting CREB vector restored CREB activation in the CA1
region of dorsal hippocampus in Tg mice
CREB activation (density of pCREB+ nuclei in the CA1 region of dorsal hippocampus) in
Tg mice microinjected with CREB vector is similar to WT mice (microinjected with GFP
or CREB vectors).
Figure S5. Increasing CREB function in the CA1 during watermaze training does
not impact distance travelled or level of thigmotaxis during training and probe test
in CaMKII∆-/- mice
(a-d) Time to platform, swim speed, distance travelled and thigmotaxis measures over the
3 watermaze training days.
(e-f) Swim speed and thigmotaxis measures during watermaze probe test.
(g) Microinjecting CREB vector into the CA1 region of dorsal hippocampus does not
rescue the spatial memory deficit of CaMKII∆-/- mice. Both αCaMKII∆-/- mice with
GFP (n=13) or CREB (n=13) vector show poor spatial memory, compared to WT
littermates microinjected with GFP (n=5) or CREB (n=5) vector. WT mice injected with
GFP or CREB spent more time in T zone compared with zones 1, 2 and 3 of the pool.
Therefore, microinjecting CREB vector into CA1 of CaMKII∆-/- mice did not rescue the
memory deficit.
(h) Microinjecting CREB vector into the CA1 region of dorsal hippocampus does not
rescue the spatial memory deficit of CaMKII∆-/- mice. Both αCaMKII∆-/- mice with
24
Yiu et al.,
Supplemental Data
GFP or CREB vector show poor spatial memory, compared to WT littermates
microinjected with GFP or CREB vector. WT mice microinjected with GFP or CREB
spent more time in T quadrant compared with other quadrants (O, L and R) of the pool.
Therefore, microinjecting CREB vector into CA1 of CaMKII∆-/- mice did not rescue the
memory deficit
Figure S6. The rescue of the spatial memory deficit in Tg mice by CREB was
independent of A plaque load or levels of aggregated A
Microinjection of CREB vector rescued the spatial memory deficit in Tg mice (Figure
2b). This rescue was independent of Aplaque load (a, b) or level of aggregated (c) or
soluble (d) Ain Tg mice. Specifically, increasing CREB levels did not reduce the
volume of tissue occupied by A plaques in dorsal hippocampus (a) or frontal cortex (b)
of Tg mice.
(c) Tg mice show high levels of aggregated Ain the dorsal hippocampus compared to
WT littermate mice. Microinjecting CREB vector does not change this.
(d) Tg mice show high levels of soluble monomeric Ain the dorsal hippocampus
compared to WT littermate mice. Microinjecting CREB vector does not change this.
Figure S7. Assessing the level of infection produced by viral vectors
(a) Examples of robust and sparse vector infection (GFP, green) following microinjection
into the CA1 region of dorsal hippocampus. Counterstained with DAPI (blue).
(b-c) Average total area infected (b) and percentage of CA1 region of dorsal
hippocampus infected (c) by CREB or GFP vectors was similar between groups.
25
Yiu et al.,
Supplemental Data
(d-e) Stereological counts estimating the number (d) and percentage (e) of GFP+ neurons
in CA1 region of dorsal hippocampus. Importantly, the level of infection produced by
microinjection of GFP or CREB vector in WT or Tg mice is similar.
Only those mice that showed robust bilateral infection of the CA1 region of dorsal
hippocampus (< 20% of CA1 region infected) were included in subsequent statistical
analyses.
Figure S8. Equivalent levels of GFP expression from p1005 and p1005-CREB
amplicon vectors
(a) The HSV amplicons used to express CREB and GFP (p1005-CREB amplicon) or
GFP alone (p1005 amplicon) in the neuronal morphological studies (Figure 5). In both
p1005-based amplicons, GFP expression is driven by a CMV promoter.
(b) GFP expression in HEK 293 cells incubated with equal amounts of p1005 or p1005CREB vector for 20 hr (MOI = 0.1). bottom: brightfield image of HEK 293 cells
expressing GFP.
(c) Western blot of lysates from HEK cells infected with either p1005 (GFP vector) or
p1005-CREB (CREB vector). As expected, p1005-CREB-infected cells show higher
levels of CREB protein when compared to p1005-infected cells. Importantly, there was
no different in levels of GFP between the two vectors. This indicates that exogenous
CREB does not affect the level of GFP expression (even when using a CMV promoter to
drive GFP expression). GAPDH loading controls are shown at the bottom.
26
Yiu et al.,
Supplemental Data
SUPPLEMENTAL REFERENCES
Clapcote SJ & Roder JC (2004) Survey of embryonic stem cell line source strains in the
water maze reveals superior reversal learning of 129S6/SvEvTac mice. Behav
Brain Res 152, 35-48.
Gallagher M, Burwell R & Burchinal M (1993) Severity of spatial learning impairment in
aging: development of a learning index for performance in the Morris water maze.
Behav Neurosci 107, 618-626.
Gass P, Wolfer DP, Balschun D, Rudolph D, Frey U, Lipp HP & Schutz G (1998)
Deficits in memory tasks of mice with CREB mutations depend on gene dosage.
Learn Mem 5, 274-288.
Jan A, Hartley DM & Lashuel HA (2010) Preparation and characterization of toxic Abeta
aggregates for structural and functional studies in Alzheimer's disease research.
Nat Protoc 5, 1186-1209.
Katzman R (1988) Alzheimer's disease as an age-dependent disorder. Ciba Found Symp
134, 69-85.
Lipp HP & Wolfer DP (1998) Genetically modified mice and cognition. Curr Opin
Neurobiol 8, 272-280.
Selkoe DJ (1994) Amyloid beta-protein precursor: new clues to the genesis of
Alzheimer's disease. Curr Opin Neurobiol 4, 708-716.
Tanghe A, Termont A, Merchiers P, Schilling S, Demuth HU, Scrocchi L, Van Leuven F,
Griffioen G & Van Dooren T (2010) Pathological Hallmarks, Clinical Parallels,
and Value for Drug Testing in Alzheimer's Disease of the APP[V717I] London
Transgenic Mouse Model. Int J Alzheimers Dis 2010.
West MJ, Slomianka L & Gundersen HJ (1991) Unbiased stereological estimation of the
total number of neurons in thesubdivisions of the rat hippocampus using the
optical fractionator. Anat Rec 231, 482-497.
27