APP and APLP2 are essential at PNS and CNS synapses for

transmission, spatial learning and LTP Sascha W. Weyer1#, Maja Klevanski1#, Andrea Delekate2#, Vootele Voikar3, Dorothee Aydin1,

Meike Hick1, Mikhail Filippov1, Natalia Drost1, Kristin L. Schaller6, Martina Saar1, Miriam A.

Vogt4, Peter Gass4, Ayan Samanta5, Andres Jäschke5, Martin Korte3, David P. Wolfer3, John H.

Caldwell6, and Ulrike C. Müller1*

Inventory of Supplementary Data

1. Supplementary Data

• Figure S1: APPs! scheme, survival analysis, body weight and western blot analysis

of APPs!-DM mice

• Figure S2: APPs!-DM mice are not impaired in basal locomotion and display

hyperactivity in their home cage and a novel environment

• Figure S3: APPs!-DM mice show unaltered paired pulse facilitation but reduced

RRP and reduced ability to sustain vesicle release

• Figure S4: APPs!-DM mice show no alterations in hippocampal and cortical

morphology and normal morphology of CA1 neurons

• Figure S5: APPs!-DM mice are impaired in species-typic behavior

• Figure S6: Description of experimental IntelliCage modules

• Figure S7: APPs!-DM mice show alteration in IntelliCage free adaptation

• Supplementary Text to Figure S7

• Figure S8: Behavioral alterations of APPs!-DM and APLP2-KO control mice are

independent of sex

• Figure S9: Home cage activity (cohorte 2, both sexes)

• Figure S10: Comparative transcriptom analysis of APPs!-DM and APLP2-KO

controls, with related Table 1, 2

• Figure S11: APPs!-DM mice exhibit impaired LTP in aged animals, yet normal

basal synaptic transmission and short-term plasticity.

• Table S3: Comparison of phenotypes of APP/APLP mutant mice

2. Supplemental Experimental Procedures • Supplementary scheme S1: Synthesis of ADAM10 inhibitor GI254023X

3. Supplemental References

2

Supplementary Data

Figure S1

Figure S1. APPs! scheme, survival analysis, body weight and western blot analysis of

APPs!-DM mice

(A) Scheme depicting APP structure and APPs! truncation generated by knockin

technology. APPs! knockin (KI) mice were obtained by gene targeting in ES cells (Ring et al,

2007). These mice express APPs! under control of the endogenous APP promoter. To this end a

stop codon had been introduced behind the !-secretase cleavage site into the endogenous APP

locus.

3

(B) Survival curve. A cohort of APPs!-DM mice was followed over time for survival and

compared to APLP2-KO littermate controls. Approximately 35% of the APPs!-DM mice die

during the first postnatal week and very few die post-weaning. Whereas mice with a combined

knockout for APP and APLP2 all die shortly after birth, about half of APPs!-DM mice survive

into adulthood.

(C) Body weight. Body weight plotted against age for APPs!-DM mice (red line) and APLP2-KO

controls (black line). Statistically significant body weight differences are indicated above the

abscissa by the black bar (***p<0.001, *p<0.05, Student’s t-test). The male APPs!-DM mice (C1)

show a body weight deficit, starting from postnatal week 3 and lasting until adulthood. However

female APPs!-DM mice (C2) show a less pronounced body weight loss starting from postnatal

week 1 until week 7, and no statistical significant difference was detected at later time points.

(D) Western blot analysis. Whole brain homogenate from APPs!-DM mice and APLP2-KO

controls at the age of 12 weeks were immunoblotted with antibodies against the C-terminus of

APP (C1/6.1), APLP2 (D2-II) and APLP1 (CT11). C1/6.1 binding to the APP C-terminus was

absent in APPs!-DM. APLP2 deficiency in the APPs!-DM animals could be confirmed using the

polyclonal antibody D2-II. No compensatory up-regulation of APLP1 in the APPs!-DM animals

could be observed using CT11 antibody.

(E) Aneural cultures of C2C12 cells secrete high amounts of APPs! upon differentiation

into myotubes. C2C12 myoblasts were switched from growth medium (GM) to differentiation

medium (DM). Conditioned medium was collected at indicated time points. Upper panel:

Western blot analysis of cell lysates of undifferentiated myoblasts or differentiated myotubes

using 22C11 antibody directed against an N-terminal APP epitope. Lower panel: Western blot

analysis of supernatants using the m3.2 antibody specifically detecting secreted APPs!. Note that

upon differentiation APP expression is upregulated, which leads to the secretion of high amounts

of APPs!.

4

Figure S2

Figure S2. APPs!-DM mice are not impaired in basal locomotion and display hyperactivity

in their home cage and a novel environment.

As a baseline for subsequent cognitive tests, we also assessed basal locomotor activity and

exploratory behavior:

(A) Home cage activity. Dark and light phase home cage activity (of individually housed mice)

averaged over 5 days of recording after 24h of habituation. APPs!-DM mice were strongly

hyperactive during the dark phase. During the light phase by contrast, they were significantly less

active than APLP2-KO mice (genotype F1,20=9.3 p<0.0063, phase F1,20=112.3 p<0.0001, phase

x genotype F1,20=15.7 p<0.0008). Number of animals tested (A-C): APPs!-DM n=10, APLP2-

KO mice n=14, graphs represent data pooled from two independent cohorts of female mice tested

independently.

(B) Open field, locomotion. In order to assess their exploratory behavior in a novel environment,

APPs!-DM mice were tested in a large circular open field for 10 min on two consecutive days and

the distance moved (normalized to 1 min) was plotted. APLP2-KO mice showed the expected

habituation with activity decreasing over time. APPs!-DM mice, by contrast, showed a clearly

aberrant pattern with activity strongly increasing over time. They were also on average more

active, especially those of cohort 1 (Genotype F(1,19)=7.0 p<0.0161, genotype x cohort

F(1,19)=8.9 p<0.0077, time F(3,57)=7.6 p<0.0002, time x genotype F(3,57)=24.3 p<0.0001).

Neither velocity (APPs!-DM: 0.232 ±0.016 m/s, APLP2-KO: 0.205 ±0.004 m/s, F(1,21)=3.893

p<0.0618) nor acceleration of APPs!-DM mice were reduced (APPs!-DM: 0.209 ±0.009 m/s2,

APLP2-KO: 0.210 ±0.005 m/s2, F(1,21)=0.005, ns) during movement bouts in the open field and

5

they did not spend more time resting than APLP2-KO (APPs!-DM: 10.343 ±1.478%, APLP2-

KO: 14.081 ±1.681%, F(1,21)=2.393, ns).

(C) Open field, time in zone, expressed as % of chance level. Time spent in the center,

transition and wall zone of the open field expressed as % of chance level of the respective zone.

APPs!-DM mice lacked the strong avoidance of the center field normally observed in mice (zone

F(2,38)=69.9 p<0.0001, zone x genotype F(2,38)=25.9 p<0.0001; zone split by genotype: APLP2-

KO F(2,24)=172.7 p<0.0001, APPs!-DM F(2,14)=2.9 ns).

6

Figure S3

Figure S3. APPs!-DM mice show unaltered paired pulse facilitation but reduced RRP and

reduced ability to sustain vesicle release.

(A, B) Synaptic facilitation is the same for APLP2-KO control and APPs!-DM mice. A pair of

stimuli was given to the nerve with a variable delay (20 to 200 ms) between the first (P1) and

second (P2) stimulus.

(A) Examples for 20 ms delay from APLP2-KO control (upper, black) and APPs!-DM (lower,

red) mice. Each test consisted of 50 pairs of stimuli, only one of which is shown for each

7

genotype, and the responses were averaged. Facilitation was calculated as the average amplitude

of the second response divided by that of the first response (P2/P1). Scale bars apply to both

traces.

(B) Facilitation plotted as a function of delay between the two stimuli. None of the differences

between the APLP2-KO and APPs!-DM muscles at any time delay was statistically significant

(20 ms: p = 0.87; 30 ms: p=0.08; 50 ms: p = 0.37; 100 ms: p=0.5; 200 ms: p=0.9). The data points

at 200 ms overlap. Number of fibers: 8-17 (APLP2-KO control) and 14-31 (APPs!-DM). Number

of muscles: 4 (APLP2-KO) and 8 (APPs!-DM). Note the break in the vertical axis.

(C) Neuromuscular junctions in APPs!-DM mice have a severely reduced readily releasable

pool (RRP) of presynaptic vesicles. Response of a control fiber (black squares) and APPs!-DM

fiber (red squares) to a 20 Hz stimulus for 2 seconds. Quantal content for each response is plotted

against the cumulative number of vesicles released. Examples shown were chosen to illustrate the

average RRP of 12 fibers, 3 muscles (APLP2-KO) and 24 fibers, 3 muscles (APPs!-DM). Dashed

lines are a linear fit to the initial rapidly declining phase of release; extrapolation of this line to its

intercept with the x-axis is the estimated RRP size (maximum number of vesicles released if there

were no replenishment of the docked vesicles at active zones). Total number of vesicles released

during the two second stimulus train by the control synapse (1610 vesicles, black arrowhead) was

almost 2.5 fold greater than for the APPs!-DM synapse (652 vesicles, red arrowhead).

(D, E) Rundown of evoked release during a 40 Hz stimulus for 10 seconds. (D) Examples of

average responses for an APLP2-KO fiber (upper, black) and APPs!-DM fiber (lower, red).

Arrowhead at start of stimulus and filled circle at end of stimulus. The response during the first

and last second are shown (stimulation continued during the 8 second gap in the figure). The

endplate potentials were measured at 1, 2, 4, 6, 8, and 10 sec by averaging groups of five

responses occurring during a 100 ms window centered upon each time point and are plotted in the

following panel.

(E) Decrease in evoked postsynaptic response during a 10 s, 40 Hz train of action potentials in the

phrenic nerve. Amplitudes were normalized for APLP2-KO controls (black squares, n=12 fibers, 3

muscles) and APPs!-DM (red squares, n=24 fibers, 3 muscles). Error bars (SEM) are smaller than

the symbol for some data points. Differences at each time point (1-10 seconds) were statistically

significant (p<10-4).

8

Figure S4

Figure S4: APPs!!-DM mice show no alterations in hippocampal and cortical morphology

and normal morphology of CA1 neurons.

9

(A-B) Nissl stain shows a normal morphology of the hippocampus (CA1, CA3 and Gyrus

dentatus) and a regular cortical layering (C-D). (E-F) Calbindin immunoreactivity revealed

normal Mossy fiber projections to CA3. (G-H) Parvalbumin staining in the CA1 area shows an

unaltered pattern of GABAergic interneurons. (I-K) Dendritic morphology of CA1 hippocampal

neurons. (I) Neurolucida reconstruction of CA1 pyramidal neurons in organotypic hippocampal

slice cultures expressing farnesylated EGFP under control of the neuron specific synapsin

promoter (upper panel: max. projection of confocal images; lower panel: Neurolucida based 3D-

reconstructed neurons). Sholl analysis performed by plotting the number of intersections against

the distance from the cell body of the CA1 apical dendrites revealed no significant changes in

dendritic complexity indicated by the total crossing number (J) (p=0.2082) and number of

intersections (K). All data represent mean ±SEM (n=36 hippocampal CA1 neurons from 8 APP APPs!-DM mice; n=36 hippocampal CA1 neurons from 10 APLP2-KO mice). Scale bar: A-F =

100µm; G-I = 50µm.

10

Figure S5

Figure S5. APPs!-DM mice are impaired in species-typic behavior.

(A) Nesting. Nest building score after 24h, scale 1-5. APPs!-DM (red bar) were strongly impaired

(genotype F(1,20)=71.6 p<0.0001). Number of animals tested (A,B): APPs!-DM n=10, APLP2-

KO n=14; graphs represent data pooled from two independent cohorts of female mice tested

independently.

(B) Burrowing. Pellets removed after 4h and 24h in the burrowing test, max. to be removed =

350g. APPs!-DM (red circles) had removed much less pellets than APLP2-KO mice at both time

points (genotype F(1,20)=63.1 p<0.0001, time F(1,20)=66.3 p<0.0001, time x genotype

F(1,20)=7.0 p<0.0158).

11

Figure S6

Figure S6. Description of experimental IntelliCage modules

(A) Free adaptation. The mixed groups of wild type and mutant mice were released in the

IntelliCage with all doors opened in the corners. During adaptation the mice could habituate to the

novel environment and learned to visit the corners for obtaining water.

(B) Nosepoke adaptation. In the beginning of this phase all doors were closed and first nosepoke

of every visit opened the door for 5 seconds. In order to drink more the mice had to leave the

corner and enter again any corner.

(C) Adaptation to drinking sessions. This phase was similar to the previous one but water was

available only in two 1-hour sessions during the dark phase (starting 3 and 8 hours after beginning

of dark phase, respectively). Thus, the mice were on water deprivation schedule and developed an

increased motivation to visit the corners during specified sessions. The following learning tasks

were carried out in drinking sessions, where the percentage of correct visits provides a measure of

learning.

(D) Corner preference learning. One correct corner where the doors could be opened during the

sessions was randomly assigned for each animal (3-4 mice at each corner). The nosepokes in the

other 3 corners were not rewarded.

12

Corner preference reversal. The correct corner was changed to be the opposite of the previously

learned one.

(E) Patrolling. In this phase the mice had to learn the sequential order of visits – after each correct

visit with nose poke, the position of correct corner was rotated clockwise by one step.

13

Figure S7

Figure S7. APPs!-DM mice show abnormal behavior during free adaptation in IntelliCage

(see also Supplementary Text for Figure S7)

(A) IC early adaptation, visits. Frequency of visits during the first 8h of free adaptation in

IntelliCage. Whereas APLP2-KO control mice (black circles) showed initial exploration followed

14

by habituation, APPs!-DM mice (red circles) failed to show habituation (genotype F(1,17)=3.6

p<0.0732, time F(2,34)=2.542 p<0.09, time x genotype F(2,34)=13.1 p<0.0001; time split by

genotype: APPs!-DM F(2,14)=2.5 ns, APLP2-KO F(2,20)=30.1 p<0.0001). Number of animals

tested (A-E): APPs!-DM n = 9, APLP2-KO n = 12, graphs represent data pooled from two

independent cohorts of female mice tested independently.

(B) IC early adaptation, corner latency. Latencies to visit 1, 2, 3, and all of 4 corners for the

first time were increased ~10-fold in APPs!-DM mice compared to control mice (genotype

F(1,17)=25.7 p<.0001, corner F(3,51)=35.2 p<0.0001, genotype x corner F(3,51)=1.1 ns).

(C) IC free adaptation, visits. Visit frequency during the light and dark phase of days 2-9 of free

adaptation. APPs!-DM mice were more active during the dark phase and showed almost no

activity during the light phase (genotype F(1,17)=3.6 p<0.0743, phase F(1,17)=153.9 p<0.0001,

phase x genotype F(1,17)=15.7 p<0.0010).

(D) IC free adaptation, visit sequences. Visit sequences during days 2-9 of free adaptation:

clockwise (cw), repeated visit to same corner, anticlockwise (acw), opposite corner. Chance =

25%. APPs!-DM mice made twice as many repeated entries to the same corner than APLP2-KO

mice (sequence F(3,51)=8.9 p<0.0001, sequence x genotype F(3,51)=67,9 p<0.0001).

(E) IC free adaptation, visits to corners. Visits to the most, second, third, and least preferred

corner during days 2-9 of free adaptation. APPs!-DM mice of both cohorts showed more

pronounced avoidance of and preference for particular corners than APLP2-KO (corner

F(3,51)=80.9 p<0.0001, corner x genotype F(3,51)=27.7 p<0.0001).

Supplementary text to Figure S7 During the first hours in IC, APPs!-DM mice showed delayed exploration of the corners (Figure

S7A) and took longer than APLP2-KO to visit all corners of the cage for the first time (Figure

S7B), reminiscent of the deficits in the open field (Figure S2B).

Starting with the second day, APPs!-DM mice made more corner visits during the dark phase and

almost none during the light phase (Figure S7C), reminiscent of the pattern in individual home

cages (Figure S2A). APPs!-DM mice unlike APLP2-KO controls did not avoid reentering the

corner they had just visited (Figure S7D) and developed abnormally strong preferences for

particular corners (Figure S7E).

It is noteworthy that deficits of APPs!-DM mice during early adaptation to the cages,

hyperactivity during the dark phase, lack of corner alternation, excessive spontaneous corner

preferences, as well as in the corner preference-reversal and patrolling task are very similar to

those seen in mice with hippocampal lesions (Colacicco et al, 2009).

15

Figure S8

Figure S8: Behavioral alterations of APPs!-DM and APLP2-KO control mice are

independent of sex

(A) Rotarod. APPs!-DM mice were impaired independently of sex (genotype F(1,14)=17.1

p<0.0010, genotype x sex F(1,14)=0.6 ns, trial F(4,56)=5.5 p<0.0008, trial x genotype F(4,56)=0.2

ns, trial x genotype x sex F(4,56)=0.4 ns).

16

Number of animals tested (A-F): APPs!-DM n=7 (females: 4, males: 3), APLP2-KO n=11

(females: 7, males: 4)

(B) Grip test. APPs!-DM mice were strongly impaired during both sessions independently of sex

(genotype F(1,14)=119.2 p<0.0001, genotype x sex F(1,14)=0.4 ns, session F(1,14)=0.1 ns,

session x genotype F(1,14)=0.04 ns, session x genotype x sex F(1,14)=0.04 ns).

(C) Cage lid hang test. All APLP2-KO mice were able to hang for 60 s from an inverted cage lid.

Independently of sex, APPs!-DM fell down either immediately or within less than 10 s (Mann-

Whitney genotype U=77 p<0.0001).

(D) Open field, locomotion. Instead of showing habituation, APPs!-DM mice of either sex

increased their locomotor activity across the two sessions of the day. APPs!-DM males also

increased their activity massively from one day to the next (genotype F(1,14)=3.9 p<0.0684, sex

F(1,14)=1.5 ns, genotype x sex F(1,14)=5.009 p<0.0420, time F(3,42)=10.1 p<0.0001, time x

genotype F(3,42)=19.0 p<0.0001, time x genotype x sex F(3,42)=5.2 p<.0037).

(E) Open field, time in zone, expressed as % of chance level. Independently of sex, APPs!-DM

mice showed strongly reduced avoidance of the center field (zone F(2,28)=51.5 p<0.0001, zone x

genotype F(2,28)=11.3 p<0.0002, zone x genotype x sex F(2,28)=0.6 ns; zone split by genotype:

APLP2-KO F(2,18)=154.2 p<0.0001, APPs!-DM F(2,10)=3.1 p<0.0908).

Animals were tested in two cohorts: cohort 1 included only female mice whereas cohort 2 was

composed of both genders.

(F) T-maze alternation. % alternations in 6 trials in the T-maze spontaneous alternation task,

chance level 50%. APPs!-DM mice (red bar) were strongly impaired independently of sex.

APPs!-DM n=7 (females: 4, males: 3), APLP2-KO n=11 (females: 7, males: 4)

(G) Nesting: Comparison of females and males of cohort 2 showed that this effect was

independent of sex (genotype F(1,14)=53.8 p<0.0001, genotype x sex F(1,14)=1.0 ns) APPs!-DM

n=7 (females: 4, males: 3), APLP2-KO n=11 (females: 7, males: 4).

(H) Burrowing: Comparison of females and males of cohort 2 revealed that this effect was

present in both sexes and even larger in males (genotype F(1,14)=48.9 p<0.0001, genotype x sex

F(1,14)=7.4 p<0.0166, time F(1,14)=16.6 p<0.0011, time x genotype F(1,14)=4.7 p<0.0482, time

x genotype x sex F(1,14)=0.008 ns; genotype split by sex: F F(1,9)=17.0 p<0.0026, M F(1,5)=17.0

p<0.0042) APPs!-DM n=7 (females: 4, males: 3), APLP2-KO n=11 (females: 7, males: 4).

17

Figure S9

Figure S9: Home cage activity, comparison of male and female mice

(AB). Home cage activity during dark and light phase (of individually housed mice) averaged over

5 days of recording after 24h of habituation. APPs!-DM mice were strongly hyperactive during

the dark phase with the effect tending to be even stronger in males, which unlike females were

also hyperactive during the light phase (genotype F1,14=16.1 p<0.0013, phase F1,14=74.8

p<0.0001, phase x genotype F1,14=11.5 p<0.0044, genotype x sex F1,14=4.1 p<0.0615, phase x

genotype x sex F1,14=0.7 ns).

18

Figure S10

Table S1: APLP2-KO vs. APPs!-DM (cortex) Probe Set ID Entrez ID Gene symbol Gene name Score Fold Change q-value

1420621_a_at 11820 App amyloid beta (A4) precursor protein -15.82 0.04 0

1440153_at 11820 App amyloid beta (A4) precursor protein -14.74 0.11 0

1427442_a_at 11820 App amyloid beta (A4) precursor protein -9.09 0.32 0

Table S2: APLP2-KO vs. APPs!-DM (hippocampus) Probe Set ID Entrez ID Gene symbol Gene name Score Fold Change q-value

1420621_a_at 11820 App amyloid beta (A4) precursor protein -40.45 0.04 0

1440153_at 11820 App amyloid beta (A4) precursor protein -19.35 0.09 0

1427442_a_at 11820 App amyloid beta (A4) precursor protein -15.6 0.27 0

1420329_at 76916 4930455C21Rik RIKEN cDNA 4930455C21 gene 5.68 1.85 0

1455973_at 626870 Gm11992 predicted gene 11992 5.14 1.35 0

Figure S10: Comparative transcriptom analysis of APPs!!-DM and APLP2-KO controls.

Hippocampus and prefrontal cortex of APPs!-DM (n=3) and APLP2-KO (n=3) animals that

underwent radial maze testing were dissected (age: 10 months) and subjected to transcriptome

analysis. Note that APP is not part of the indicated number of genes. (B,C) Significance analysis

of microarrays (SAM) revealed no significant transcriptional changes between the genotypes

either in cortex (Table S1) or in hippocampus (Table S2). (D) As expected, numerous

differentially expressed genes were detected by comparing cortical and hippocampal tissue of the

same genotype.

Figure S11

Figure S11: APPs!-DM mice exhibit impaired LTP in aged animals, yet normal basal

synaptic transmission and short-term plasticity.

Schaffer collaterals of acute hippocampal slices of 10-13 month old APPs!-DM mice (red

circles), APLP2-KO control mice (black circles) or wild type (WT) mice (open circles) were

20

stimulated at 0.1 Hz (baseline) and fEPSPs recorded in CA1. Data points are averaged over 6

time-points, mean baseline slope was set to 100%.

(A) LTP of fEPSP was induced by application of TBS after 20 min baseline stimulation

(arrowhead). 60 minutes after TBS a significant difference between APPs!-DM mice and

APLP2-KO mice could be observed (p=0.001, t-test) indicated by the asterisk, WT and

APLP2-KO controls were indistinguishable from each other at that time point (APPs!-DM:

133 ±4.4%; APLP2-KO controls: 157 ±6.7%; WT: 159 ±11.3%).

(B) PPF in all tested slices showed no significant differences between genotypes.

(C) Input-output-strength revealed no alterations between APPs!-DM, APLP2-KO, and WT

mice.

(D) In organotypic hippocampal cultures LTP of fEPSP was induced by application of TBS

after 10 min of baseline recording (arrowhead). Treatment of APP-KO cultures for 5 days

with the !-secretase (ADAM10) inhibitor (dark green circles) lead to lower LTP induction

rates after TBS, as compared to DMSO treated controls (light green circles). APP-KO plus

0.01% DMSO (control): 120.7 ±8.1% (35-50min after TBS), n=8; APP-KO with GI254023X

in DMSO: 102.3 ±7.9%, n=9, (p=0.02 (Two-way ANOVA, 11-40min, these are all timepoints

after TBS); p=0.069, t-test; at t=35-40min)

Table S3: Comparison of phenotypes of APP/APLP mutant mice

(a) this study (b) Ring et al, (2007) (c) Wang et al, (2005) (d) von Koch et al, (1997) (e) Zheng et al, (1995) (f) Magara et al, (1999) (g) Heber et al, (2000) (h) Seabrook et al, (1999) (i) (Phinney et al, (1999) (j) Schrenk-Siemens et al, (2008) (k) Yang et al, (2005) (l) Yang et al, (2007)

Phenotype

APPs!-KI

(b)

(adult)

APP-KO (b, e, f)

(adult)

APLP2-KO (a, d, g)

(adult)

APPs!-DM

(a)

(adult)

APP/APLP2-DKO

(P0)

Viability, gross morphology

- Viable, - Normal

- Viable, - Growth deficit

- Viable, - Normal

- majority of animals survive, - Growth deficit - Lethal within 24 h after birth (d, g))

NMJ morphology

n.d. Normal Normal

- Widening of endplate band - Excessive secondary nerve branching - AChR area reduced - Synaptophysin area reduced, - subtle reduction in AChR/Syn colocalization - Impaired postsynaptic maturation and/or maintenance: fragmented and plaque-like endplates in adult mice

- Widening of endplate band (c) - Excessive secondary nerve branching (c) - Pronouced reduction in AChR/Syn colocalization (c) - Reduced density of synaptic vesicles (reduced active zone size) (k)

NMJ electrophysiology

Normal - Reduced PPF (l) Normal

Spontanous responses: - Subtle defect in sponatous NT release:Altered frequency distribution with reduced average frequency, increased amplitude Evoked responses: - Reduced quantal content - Reduced RRP size - Impairment of sustained release

Spontanous responses: Severe defect in spontanous NT release (c) - 50% of fibers lack detectable minis Evoked responses: Severe defect in evoked NT release (c) - 25% of fibers lack evoked responses

PNS

Motor behavior

Normal Subtle grip strength deficit Normal

- Pronounced grip strength deficit and - Impairment in Rotarod and cage lid hanging - No impairment in basal locomotion - Abnormal home cage activity: hyperactive during dark phase

Not applicable at P0

Brain morphology Normal

- Subtle alterations in brain morphology: reduced brain weight and forebrain commissures (f) - Unaltered synaptic density (i)

Normal - Normal: unaltered synaptic density in Hippocampus - Unaltered dendritic morphology of CA1 neurons, - Unaltered spine density (apical dendrites of CA1 neurons)

Normal at P0

Basal synaptic transmission Normal Normal Normal Normal

Defect in basal synaptic transmission in organotypic hippocampal cultures (input-output curves) (j)

Short term plasticity Normal Normal Normal Normal n.d.

Long term plasticity Normal Impaired LTP in aged mice (age:9-12 months) (b, i) Normal

- Impaired LTP already in young mice (age:3-5 months) - Inhibition of GABAA-R rescues LTP deficit

n.d.

CN

S

Hippocampus dependant behavior

Normal (Morris water maze)

Only aged mice show impaired spatial learning and memory (Morris water maze) (b, j)

Normal

- Severe impairments already in young mice - Impaired working memory (T-maze) - Impaired spatial learning and short term memory (radial maze, IntelliCage operant conditioning) - Highly abnormal species typic behaviors (nesting test, burrowing test)

Not applicable at P0

Supplemental Experimental Procedures Mouse crosses and Genotyping The generation of APLP2-KO mice (von Koch et al, 1997) and APPs! knockin (APPs!-KI)

mice was described previously (Ring et al, 2007). Analysis of survival rates, body weight and

electrophysiological recordings at the NMJ were performed with mice of mixed

(129xC57BL/6) genetic background. For all behavioral experiments (motor behavior, grip

strength, home cage activity, species typic behaviors, tests for learning and memory), as well

as NMJ and CNS morphology, CNS electrophysiology including LTP measurements,

Western blot analysis and Microarray analysis we used mice that had been backcrossed six

times to C57BL/6 mice. APPs! double mutants (APPs!/s!-KI/ APLP2-KO, termed APPs!-

DM) and the corresponding APLP2-KO littermate controls were generated by three

consecutive crosses. Single mutants of APPs!-KI and APLP2-KO were inter-crossed, and

mice heterozygous for both loci were backcrossed to APLP2-KO single mutants to obtain

25% offspring homozygous knockout for APLP2 and heterozygous for APPs!. These mice

[APPs!/wt/APLP2-KO] were further inter-crossed to obtain APPs!-DM [APPs!/s!/APLP2-KO]

animals and their APLP2-KO littermates. For genotyping the same primers were used as in

Ring et al. 2007.

Survival rates and body weight

Mice from heterozygous intercrosses [APPs!/wt x APPs!/wt], both on an APLP2-deficient

background, were marked at birth. The genotype was analyzed at birth and confirmed again 4

weeks later (or at the time point of death). We have calculated the deviation from Mendelian

segregation using Chi-square statistics. Starting from a total number of 143 pups, we found

that the APPs!-DM mice were born at a normal Mendelian ratio (Chi-square F(2)=1,4056, ns;

APPs!-DM [APPs!/s!/APLP2-KO] n=41, Het mice [APPs!/wt/APLP2-KO] n=65, controls

[APPwt/wt/APLP2-KO] n=37) suggesting no loss of APPs!-DM pups during embryonic

development. The body weight was measured weekly and followed for 11 weeks for the

APPs!-DM mice and their respective APLP2-KO littermate controls.

Tissue processing and Western blot analysis

Western blot analysis of whole-brain homogenates of 12-weeks-old APPs!-DM and APLP2-

KO animals was performed as previously described (Heber et al, 2000). Proteins (20 "g/lane)

were separated on 8% TRIS-Tricine gels and electrotransferred to PVDF membrane. After

blocking, the blot was incubated overnight with the primary antibody followed by a 1h

incubation with horseradish-coupled secondary antibody. Western blot signals were detected

by enhanced chemiluminescence (Super Signal West Pico, Pierce, Rockford, IL). Antibodies

used for Western blots: Specific antibodies directed against the APP C-terminus (C1/6.1,

23

1:5000), APLP1 (CT11, 1:10000, Calbiochem, La Jolla), APLP2 (D2-II, 1:10000,

Calbiochem), and ß-tubulin (MAB3408, 1:10000, Chemicon) were used.

Histology, neuromuscular junction

Monitoring of nerve branching and quantification of synapse distribution

Stacks of confocal images from the S2 region obtained using a 10# objective (air, N.A. 0.45)

were projected into one plane and joined to an overview image. Subsequently, the color

channels were split. For morphological analysis a 2485 "m high region of 2 months old mice

(3 diaphragms per genotype) starting 1491 "m ventral to (towards the sternum) the entry point

of the phrenic nerve was selected. The coordinates of individual synapses were determined

with the help of ImageTool (UTHSCSA) software. A least squares linear fit regression line

was plotted and superimposed on the scatter plot of the data points. To calculate the area

covered by synapses the selected area was divided into 10 stripes of equal height, oriented

perpendicularly to the regression line. Area was calculated within each stripe as a product of

height and width defined by the most distal synapses within the stripe. Total area is a sum of

the 10 individual rectangles.

For distributional analysis the synapse band region of 2 months old mice was divided into 7

adjacent strips of 178 "m width, oriented parallel to the regression line with stripe 4 being

centered on the regression line. The number of synapses within each stripe was determined

and plotted. Data were collected from 3 diaphragm muscles per genotype.

Quantification of neuromuscular synaptic structure

Image stacks of individual synapses from the S3 region of the diaphragm were obtained using

a 60# objective (oil, N.A. 1.4) and merged into maximal projection images. From each

diaphragm (3 mice per genotype) 30-38 synapses were examined. Synaptophysin-AChR

colocalization coefficient was calculated as a quotient of synaptophysin covered area divided

by area occupied by AChRs. Degree of fragmentation was obtained by analyzing binarized

images of individual synapses using Particle Analyzer tool (ImageJ).

24

Morphological classification of neuromuscular synapse

Classification analysis was performed with 54-78 synapses per mouse (3 mice per genotype).

Synapses were classified into 3 categories. The pretzel class includes synapses composed of

1-3 fragments excluding plaque-like structures. Synapses composed of $ 4 fragments are

defined as fragmented. Plaque-like synapses were identified as oval/half-moon-shaped,

lacking complex branching. Note also the lack of sharp contours with high AChR density.

NMJ Electrophysiology

The diaphragm muscle with 5-10 mm of the attached phrenic nerve was dissected from adult

mice (7-8 weeks old), mounted in a Sylgard-lined dish, and superfused with oxygenated (95%

O2, 5% CO2) modified Tyrode’s solution (in mM: 125 NaCl, 5.37 KCl, 24 NaHCO3, 1 MgCl2,

1.8 CaCl2, 11 glucose, pH 7.4) for at least 1h before recording. Recordings were from the left

hemi-diaphragm in region S2. A tight-fitting glass suction electrode was used for nerve

stimulation (stimulus duration 0.1 ms, amplitude 0.1-1 Volt). Intracellular muscle fiber

recordings were made at room temperature (20-22 °C) with 20-30 M% resistance glass

microelectrodes filled with 3M KAc. Muscles were paralyzed with µ-conotoxin (1 µM;

Bachem, Torrance, CA, USA) to block skeletal muscle but not axonal voltage-gated sodium

channels. Data were acquired with custom written Matlab (Mathworks, Natick, MA, USA)

programs and digitized at 5 KHz with DAQCard-1200 (National Instruments). Data were

analyzed with Clampfit 9 (Molecular Devices) and Origin 6.1 (OriginLab). Endplate

potentials (EPPs) and quantal content were corrected for nonlinear summation (McLachlan

and Martin, 1981). Endplate potentials were not corrected for extracellular field potentials

sometimes generated by neighboring muscle fibers. Spontaneous activity was typically

recorded for one minute except for those fibers with very low spontaneous release, for which

recordings were made for 2-3 minutes. Quantal content was calculated from the average of

50-100 EPPs at a stimulation rate of 0.5 Hz. Because of the pulse-to-pulse variability in the

EPP amplitude during trains of 20 and 40 Hz stimuli, the EPP amplitude during rundown

experiments (Figure 2H and S3D) was calculated as the average of five EPPs closest to each

data point (equivalent to an average response within a window duration of 200 ms (20 Hz)

and 100 ms (40 Hz)). All values are expressed as the mean ±SEM. Statistical significance was

evaluated with Student’s t-test.

25

CNS Histology

Quantitative analysis of synaptic puncta within the hippocampus

Quantification of synaptic puncta in striatum radiatum of hippocampal CA1 region. Five

microscopic fields (of 110 "m x 110 "m) per slice from 18 slices (3 slices per animal, 3

animals per genotype) were imaged with a laser-scanning confocal microscope (Nikon A1R)

using a 60# objective (oil, N.A. 1.4). Maximum projections of confocal z-stacks were

performed and synaptic puncta were counted manually for 2 slices. A comparable threshold

was set and used for all slices. The number of synaptophysin-positive puncta was obtained

using ImageJ software. Data obtained from APPs!-DM were normalized to APLP2-KO mice.

Preparation of hippocampal slice cultures for hippocampal neuron morphology analysis

Organotypic hippocampal slice cultures were prepared as previously described (Stoppini et al,

1991). P0 mice were decapitated, the hippocampi were dissected in ice-cold Gey’s Balanced

Salt Solution (GBSS) and sliced transversely at a thickness of 400"m on a tissue chopper

(McIllwain, Wood Dale, IL). The slices were plated onto Millicell-CM membrane inserts

(Millipore, Bedford, MA) and cultivated in a 37°C, 7% CO2, 99% humidity incubator. To

reduce the number of non-neuronal cells, antimitotic drugs (uridine, cytosine-&-D-

arabinofuranoside hydrochloride, and 5-fluoro-2’-deoxyuridine) were applied for 24 h 3 days

after preparation.

Bioistic transfection

Organotypic hippocampal slice cultures (OTCs) were transfected at DIV14 using the Helios

Gene Gun system (Bio-Rad). Plasmid coated gold microcarriers were shot onto the slices

using a helium burst of 70 PSI. To avoid tissue damage, culture inserts with a pore width of 3

"m were used as filters. Bullets for transfection were prepared according to the

manufacturer’s instructions (BioRad). Briefly, 2"g plasmid DNA/mg gold was coated onto

0.6"m gold microcarriers. To visualize neuronal morphology in detail, a membrane targeted

(farnesylated) form of EGFP under control of the neuron specific synapsin promoter was

transfected. For coating of DNA onto the gold microcarriers, CaCl2 precipitation was

performed (Wellmann et al, 1999; O’Brien & Lummis, 2006). OTCs were fixed with PFA

(4%) 3d post-transfection.

26

Transcriptom analysis:

Animals

All animals were adult females (38-40 weeks of age). The same set of APPs!-DM and

APLP2-KO animals were used that underwent radial maze testing.

RNA preparation and microarray data generation

Animals were sacrificed by cervical dislocation. Mouse brains were dissected and stored in

RNAlater (Qiagen) at -20°C. Subsequently, the prefrontal cortex and hippocampus were

dissected and used for total RNA preparation (RNAeasy kit, Qiagen). The quality of RNA

was assessed with a spectrophotometer and Bioanalyzer (Agilent). 1µg of total RNA was used

for cDNA preparation (Oligo dT method, Invitrogen). Subsequent cRNA was prepared with

Affymetrix One-Cycle Target Labeling and Control Reagent kit (Affymetrix Inc., Santa

Clara, California, USA). The biotinylated cRNA was hybridized onto GeneChip Mouse

Genome 430 2.0 Arrays (Affymetrix, Santa Clara). Chips were washed and scanned on the

Affymetrix Complete GeneChip® Instrument System generating digitized image data files.

Raw and processed data discussed in this publication have been deposited in the

NCBI's Gene Expression Omnibus database (GEO) and are accessible through GEO

Series accession number GSE27888.

(<http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE27888)

Statistical analysis

Data analysis and processing were carried out within the statistical computing environment R,

version 2.8.0, using Bioconductor, BioC Release 2.4 (Gentleman et al, 2004).

The data were processed with the RMA algorithm (Robust Multiarray Average) developed by

Irizarry et al. (Irizarry et al, 2003) and normalized using quantile normalization (Bolstad et al,

2003). Significant differentially expressed probesets were detected by a Significance Analysis

of Microarrays (SAM) (Tusher et al, 2001). As a cut-off value for significance, the false

discovery rate (FDR) was set to 0%.

CNS electrophysiology, Slice preparation

In brief, mice were anesthetized and decapitated; the brain was quickly transferred into ice-

cold carbogenated (95% O2, 5% CO2) artificial cerebrospinal fluid (ACSF). Hippocampi were

cut with a vibratome (400 "m; VT 1000S; Leica, Nussloch, Germany). The ACSF used for

electrophysiological recordings contained 125.0 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4,

2 mM MgCl2, 26 mM NaHCO3, 2.0 mM CaCl2, 25 mM glucose. Recordings were done in a

submerged recording chamber at 32°C.

27

CNS electrophysiological recordings

After placing the slices in a submerged recording chamber, field excitatory postsynaptic

potentials (fEPSPs) were recorded in the stratum radiatum of the CA1 region with a glass

micropipette (resistance 3-15 M!) filled with 3 M NaCl at a depth of "150-200 "m.

Monopolar tungsten electrodes were used for stimulating the Schaffer collaterals at a

frequency of 0.1 Hz. Stimulation was set to elicit a fEPSP with a slope of "40-50% of

maximum for LTP recordings. After 20 min baseline stimulation, LTP was induced by

applying theta-burst stimulation (TBS), in which a burst consisted of 4 pulses at 100 Hz

which were repeated 10 times in a 200 ms interval (5 Hz). Three such trains were used to

induce LTP at 0.1 Hz.

Basic synaptic transmission and presynaptic properties were analyzed via input-output-

(IO) measurements and paired pulse facilitation. The IO-measurements were performed by

application of a defined value of current (25-250 "A in steps of 25 "A). Paired pulse

facilitation was performed by applying a pair of two stimuli in different inter-stimulus-

intervals (ISI) ranging from 10, 20, 40, 80 to 160 ms. Data were collected, stored and

analyzed with LABVIEW software (National Instruments, Austin, TX). The initial slope of

fEPSPs elicited by stimulation of the Schaffer collaterals was measured over time, normalized

to baseline and plotted as average ±SEM.

Behaviour

General: The mice were transferred to single cages before the beginning of the experimental

period and tested during the dark phase of the cycle (lights on between 8 p.m. and 8 a.m.).

Standard mouse chow, water, and nesting material were available ad libitum. The home cage

rack was brought to the test room at least 30 min before each experiment. Mice distributed

over two cohorts, were analyzed in a blinded manner in the following order of tests: cohort 1:

IC (age 3.1-4.4 mo), open field (5.0 mo), grip strength and cage lid hanging test (5.0 mo),

rotarod (5.0 mo), nesting and burrowing tests (5.3 mo), T-maze (5.5 mo), home cage activity

(5.7 mo); cohort 2: home cage activity (average age 3.6mo), open field (3-7mo), grip strength

and cage lid hanging test (3.8 mo), rotarod (3.8 mo), T-maze (4.0 mo), nesting, and burrowing

tests (4.1 mo), IC (females only, 4.4-6.0 mo), radial maze (females only, 7.8 mo).

Open field

Activity was tested as described previously (Madani et al, 2003). In brief, the open field was a

dimly lit circular arena (150 cm diameter) in which the mice were observed and tracked using

Noldus EthoVision 3.1 software (www.noldus.com) for 10 min each on two consecutive days.

The arena was divided into a wall zone (18% of surface, 7 cm wide), a center zone (50%), and

28

a transition zone in between. For in depth analysis of locomotion, recorded tracks were

segmented into discrete episodes of resting, small movements (lingering), and long distance

locomotion (Drai & Golani, 2001; Madani et al, 2003). Velocity of locomotion was estimated

by averaging velocity across all episodes of long distance locomotion. As measure of

acceleration, we divided maximal velocity of each episode of long distance locomotion by

episode duration and computed the median of this quotient across all episodes.

Calculation of velocity, acceleration, and resting time

For in depth analysis of locomotion, recorded tracks were segmented into discrete episodes of

resting, small movements (lingering) and long distance locomotion (Drai & Golani, 2001;

Madani et al, 2003). Velocity of locomotion was estimated by averaging velocity across all

episodes of long distance locomotion. As measure of acceleration, we divided maximal

velocity of each episode of long distance locomotion by episode duration and computed the

median of this quotient across all episodes.

Grip strength

Forepaw grip strength was measured as described previously (Ring et al, 2007) using a

newtonmeter (max. force: 300 g) that was positioned horizontally and attached to a metallic

ring of 5.5 cm diameter and 3 mm thickness. Mice were held by the tail and allowed to grasp

the ring with both forepaws. They were then gently pulled back until they released the ring.

Five measurements were obtained each on 2 consecutive days and averaged.

Home cage activity

Home cage activity was recorded as described previously (Madani et al, 2003) using a cage

rack equipped with one passive IR sensor per mouse (ActiviScope, New Behavior Inc., Zurich

Switzerland, www.newbehavior.com). The sensors detected any locomotion and remained

silent only when the mice were sleeping or grooming. Recording started after a habituation

period of at least 18h and circadian profiles were calculated by averaging data from at least 4

recording days.

29

Species typic behavior

Nesting test

Nest building was studied as described (Deacon, 2006a). At the beginning of the dark phase,

mice were placed in individual testing cages (Type II, 267 x 207 x 140 mm) containing

regular bedding and a Nestlet of 3 g compressed cotton (Ancare, Bellmore, NY). After 24h

the nests were assessed on a rating scale of 1-5: 1 = Nestlet > 90% intact, 2 = Nestlet 50-90%

intact, 3 = Nestlet mostly shredded but no identifiable nest site, 4 = identifiable but flat nest,

5 = crater-shaped nest.

Burrowing test

Burrowing was studied as described (Deacon, 2006b). A grey plastic tube (inner diameter 6.3

cm, length 18.2 cm) was filled with 350 g standard diet food pellets (Kliba Nafag 3430,

Provimi Kliba AG, Kaiseraugst, Switzerland, ca. 3 g each) and placed at a slight angle into a

large standard transparent mouse cage (Type III, 425 x 266 x 155 mm). The lower end of the

tube was closed, resting on the cage floor. The open end was supported 3.5 cm above the floor

by two metal bolts. The cage floor was covered with fresh standard bedding material and a

cardboard environmental enrichment tube was also placed in the test cage. Procedure. At the

beginning of the dark period, mice were placed individually in test cages and left in their

familiar animal room for an observation period of 4h after which the amount of non-displaced

food was measured. This was followed by a second observation period of 20h. Water was

available ad libitum during the entire period. It was assumed that the amount of food eaten per

mouse (2 ±0.5 g) was a very small proportion of the 350 g available and approximately equal

across the groups.

Behavior: learning and memory

Radial-maze

The working memory procedure on the 8-arm radial maze was carried out as described

previously (Lang et al, 2006). The apparatus was constructed of grey poly-vinyl chloride.

Eight arms (7 x 38 cm) with clear Perspex sidewalls (5 cm high) extended from an octagonal

centre platform (diameter 18.5 cm, distance platform centre to end of arm 47 cm). It was

placed 38 cm above the floor in a dimly lit room (4 x 40W bulbs, 12 lux) rich in salient

extramaze cues (same room as for water-maze testing). Small cereal pellets (ca. 6 mg) were

placed as baits in small metal cups (diameter 3 cm, 1 cm deep) at the end of each arm, in such

a way that the mouse could not see them without completely entering the arm. A reversed box

of clear Perspex served to confine the mouse on the centre platform before each test session

during which the mice were allowed to move freely on the maze. Mice were gradually

30

reduced to and maintained at 85% of their free-feeding body weight using a premeasured

amount of chow each day. Water was available ad libitum. Mice performed one trial per day

lasting maximally 10 min or until the animal had collected all pellets. They began with two

habituation sessions during which they were accustomed to collecting pellets from the maze

that were distributed all over the maze. During the following 10 training trials each cup was

baited only with one pellet. Behavioral measures: Consumption of each pellet was recorded

by pressing a designated key on the keyboard. With this information and the video-tracked

xy-coordinates, the following measures were computed: total duration of trial, time spent

moving, duration of arm visits, time spent in pellet area, decision time between visits, average

velocity, velocity in/out of arms, number of omitted arms, number of bait neglect errors

(failure to consume pellet in a baited arm), number of aborted visits (not reaching pellet area),

number of working memory errors (visits to already emptied arms), number of correct choices

before first error, correct choices out of first eight, relative frequency of serial choices,

relative frequency of repeated angles, relative frequency of favorite choice angle, number of

visits to favorite arm.

T-maze

Spontaneous alternation on the T-maze was assessed as described in (Deacon & Rawlins,

2006). The T-maze was made of grey PVC. Each arm measured 30 # 10 cm. A removable

central partition extended from the centre of the back goal wall of the T to 7 cm into the start

arm. This totally prevented the mouse from seeing or smelling the non-chosen arm during the

sample run, thus minimizing interfering stimuli. The entrance to each goal arm was fitted with

a guillotine door. Each trial consisted of an information-gathering, sample run, followed

immediately by a choice run. For the sample run a mouse was placed in the start arm, facing

away from the choice point with the central partition in place. It was allowed to choose a goal

arm and confined there for 30 s by lowering the guillotine door. Then the central partition was

removed, the mouse replaced in the start arm, and the guillotine door was raised. Alternation

was defined as entering the opposite arm to that entered on the sample trial (whole body,

including tail). Three trials were run per day with an inter-trial interval of approximately 60

min. Each mouse received 6 trials in total and for data analysis the percentage of correct

choices was calculated.

IntelliCage testing

For individual identification, the mice were subcutaneously injected with RFID transponders

(Datamars SA, Bedano, Switzerland). The IntelliCage (NewBehavior AG, Zurich,

Switzerland) is an apparatus designed to fit inside a large cage of 20 cm high, 55 cm long and

31

38 cm wide at the base (Tecniplast, 2000P). The apparatus itself provides four recording

chambers that fit into the corners of the housing cage covering a right-angle triangular 15 cm

x 15 cm x 21 cm area of floor space each. Access into the chambers is provided via a tubular

antenna (50 mm outer and 30 mm inner diameter) reading the transponder codes. The

chamber, equipped with a proximity sensor, contains two openings of 13 mm diameter (one

on the left, one on the right) that give access to water bottle nipples. These openings are

crossed by photo beams recording nosepokes of the mice and the holes can be closed by small

motorized doors, thus barring access to water bottles in each corner. Four triangular red

shelters (Tecniplast, Buguggiate, Italy) were placed in the middle of the IntelliCage and used

as sleeping quarters and as a stand to reach the food. The floor was covered with a thick (2-3

cm) layer of aspen bedding (5 x 5 x 1 mm, Abedd, Lab & Vet Service GmbH, Vienna,

Austria). The IntelliCage was controlled by a computer with dedicated software, executing

preprogrammed experimental schedules and registering the number and duration of visits to

the corners, nosepokes to the doors and lickings as behavioral measures for each mouse. Up

to 13 mutant and control animals were tested together in the same cage.

Synthesis and Characterization of GI254023X

Inhibitor GI254023X was synthesized in twelve steps (Supplementary Scheme S1),

essentially as described in Andrews et al. (2000) with the following modifications: (1) in the

synthesis of 1, highly carcinogenic HMPA was replaced by less hazardous 1,3-dimethyl-2-

imidazolidinone (DMEU); (2) intermediate 2 was isolated as a stable compound; (3) in the

synthesis of 4, the aqueous workup step was omitted, increasing yield to 100%.

All intermediates shown were isolated and fully characterized by 1H NMR, 13C NMR, and

mass spectrometry.

32

Supplementary Scheme S1: Synthesis of GI254023X

Analytical Data of GI254023X: 1H NMR (300 MHz, CD3OD)

d 1.00 (s, 9H), 1.17 (t, 3H) and 1.27 (d, 2H), 1.50 (m, 4H), 2.50-2.87 (m, 6H), 3.83 and 4.51

(dq, 1H), 4.27 (s, 1H), 7.11 (m, 3H), 7.21 (m, 2H), 8.00 and 8.29 (s, 1H) 13C NMR (300 MHz, CD3OD)

d 15.49, 16.54, 17.69, 26.01, 27.28, 30.03, 31.32, 35.20, 36.86, 50.20, 53.74, 58.71, 62.25,

66.92, 126.73, 129.29, 129.38, 149.51, 159.70, 164.26, 173.02, 175.83

FAB-MS

[M+3H]+ = C21H33N3O4 m/z 394.1

33

Supplemental References Andrews RC, Andersen MW, Cowan DJ, Deaton DN, Dickerson SH, Drewry DH, Gaul MD, Luzzio MJ, Marron BE, Rabinowitz MH. (2000). Preparation of peptidyl formamide compounds as therapeutic agents. WO 2000012083, Research Triangle Park Glaxo Group Ltd., 2000. Bolstad BM, Irizarry RA, Astrand M, Speed TP (2003) A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics 19: 185-193 Colacicco, G., Voikar, V., Vannoni, E., Lipp, H.-P., and Wolfer, D.P. (2009). Cognitive functions of hippocampus lesioned mice assessed in the IntelliCage. Neuroscience Meeting Planner. Society for Neuroscience, 2009, Program No. 478.19. 2009. Deacon, R.M. (2006a). Assessing nest building in mice. Nat Protoc 1, 1117-1119.

Deacon, R.M. (2006b). Burrowing in rodents: a sensitive method for detecting behavioral dysfunction. Nat Protoc 1, 118-121.

Deacon, R.M., and Rawlins, J.N. (2006). T-maze alternation in the rodent. Nat Protoc 1, 7-12.

Drai, D., and Golani, I. (2001). SEE: a tool for the visualization and analysis of rodent exploratory behavior. Neurosci Biobehav Rev 25, 409-426.

Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S, Ellis B, Gautier L, Ge Y, Gentry J, Hornik K, Hothorn T, Huber W, Iacus S, Irizarry R, Leisch F, Li C, Maechler M, Rossini AJ, Sawitzki G, Smith C, Smyth G, Tierney L, Yang JY, Zhang J (2004) Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 5: R80 Heber, S., Herms, J., Gajic, V., Hainfellner, J., Aguzzi, A., Rulicke, T., von Kretzschmar, H., von Koch, C., Sisodia, S., Tremml, P., et al. (2000). Mice with combined gene knock-outs reveal essential and partially redundant functions of amyloid precursor protein family members. J Neurosci 20, 7951-7963.

Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, Scherf U, Speed TP (2003) Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4: 249-264 Lang, U.E., Wolfer, D.P., Grahammer, F., Strutz-Seebohm, N., Seebohm, G., Lipp, H.P., McCormick, J.A., Hellweg, R., Dawson, K., Wang, J., et al. (2006). Reduced locomotion in the serum and glucocorticoid inducible kinase 3 knock out mouse. Behav Brain Res 167, 75-86.

Madani, R., Kozlov, S., Akhmedov, A., Cinelli, P., Kinter, J., Lipp, H.P., Sonderegger, P., and Wolfer, D.P. (2003). Impaired explorative behavior and neophobia in genetically modified mice lacking or overexpressing the extracellular serine protease inhibitor neuroserpin. Mol Cell Neurosci 23, 473-494.

Magara F, Muller U, Li ZW, Lipp HP, Weissmann C, Stagljar M, Wolfer DP (1999) Genetic background changes the pattern of forebrain commissure defects in transgenic mice underexpressing the beta-amyloid-precursor protein. Proc Natl Acad Sci U S A 96: 4656-4661

34

McLachlan, E.M., and Martin, A.R. (1981). Non-linear summation of end-plate potentials in the frog and mouse. J Physiol 311, 307-324.

Phinney AL, Calhoun ME, Wolfer DP, Lipp HP, Zheng H, Jucker M (1999) No hippocampal neuron or synaptic bouton loss in learning-impaired aged beta-amyloid precursor protein-null mice. Neuroscience 90: 1207-1216 O'Brien JA, Lummis SC (2006) Biolistic transfection of neuronal cultures using a hand-held gene gun. Nat Protoc 1: 977-981 Ring, S., Weyer, S.W., Kilian, S.B., Waldron, E., Pietrzik, C.U., Filippov, M.A., Herms, J., Buchholz, C., Eckman, C.B., Korte, M., et al. (2007). The secreted beta-amyloid precursor protein ectodomain APPs alpha is sufficient to rescue the anatomical, behavioral, and electrophysiological abnormalities of APP-deficient mice. J Neurosci 27, 7817-7826.

Schrenk-Siemens K, Perez-Alcala S, Richter J, Lacroix E, Rahuel J, Korte M, Muller U, Barde YA, Bibel M (2008) Embryonic stem cell-derived neurons as a cellular system to study gene function: lack of amyloid precursor proteins APP and APLP2 leads to defective synaptic transmission. Stem Cells 26: 2153-2163 Seabrook GR, Smith DW, Bowery BJ, Easter A, Reynolds T, Fitzjohn SM, Morton RA, Zheng H, Dawson GR, Sirinathsinghji DJ, Davies CH, Collingridge GL, Hill RG (1999) Mechanisms contributing to the deficits in hippocampal synaptic plasticity in mice lacking amyloid precursor protein. Neuropharmacology 38: 349-359 Stoppini L, Buchs PA, Muller D (1991) A simple method for organotypic cultures of nervous tissue. J Neurosci Methods 37: 173-182 Tusher VG, Tibshirani R, Chu G (2001) Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A 98: 5116-5121 von Koch, C.S., Zheng, H., Chen, H., Trumbauer, M., Thinakaran, G., van der Ploeg, L.H., Price, D.L., and Sisodia, S.S. (1997). Generation of APLP2 KO mice and early postnatal lethality in APLP2/APP double KO mice. Neurobiol Aging 18, 661-669. Wang P, Yang G, Mosier DR, Chang P, Zaidi T, Gong YD, Zhao NM, Dominguez B, Lee KF, Gan WB, Zheng H (2005) Defective neuromuscular synapses in mice lacking amyloid precursor protein (APP) and APP-Like protein 2. J Neurosci 25: 1219-1225 Wellmann H, Kaltschmidt B, Kaltschmidt C (1999) Optimized protocol for biolistic transfection of brain slices and dissociated cultured neurons with a hand-held gene gun. J Neurosci Methods 92: 55-64 Yang G, Gong YD, Gong K, Jiang WL, Kwon E, Wang P, Zheng H, Zhang XF, Gan WB, Zhao NM (2005) Reduced synaptic vesicle density and active zone size in mice lacking amyloid precursor protein (APP) and APP-like protein 2. Neurosci Lett 384: 66-71 Yang L, Wang B, Long C, Wu G, Zheng H (2007) Increased asynchronous release and aberrant calcium channel activation in amyloid precursor protein deficient neuromuscular synapses. Neuroscience 149: 768-778 Zheng H, Jiang M, Trumbauer ME, Sirinathsinghji DJ, Hopkins R, Smith DW, Heavens RP, Dawson GR, Boyce S, Conner MW, Stevens KA, Slunt HH, Sisoda SS, Chen HY, Van der

35

Ploeg LH (1995) beta-Amyloid precursor protein-deficient mice show reactive gliosis and decreased locomotor activity. Cell 81: 525-531