www.elsevier.com/locate/ynbdi

Neurobiology of Disease 22 (2006) 463 – 472

DYRK1A BAC transgenic mice show altered synaptic plasticity with

learning and memory defects

Kyoung-Jin Ahn,a,1 Hey Kyeong Jeong,a,1 Han-Saem Choi,b Soo-Ryoon Ryoo,a

Yeon Ju Kim,a Jun-Seo Goo,c Se-Young Choi,b Jung-Soo Han,a

Ilho Ha,a,* and Woo-Joo Songa,*

aGraduate Program in Neuroscience and Institute for Brain Science and Technology (IBST), Inje University, Daejeon 305-804, Republic of KoreabDepartment of Physiology and Dental Research Institute, School of Dentistry, Seoul National University, Seoul 110-749, Republic of KoreacDepartment of Laboratory Animal Science, College of Medicine, Inje University, Busan 614-735, Republic of Korea

Received 30 September 2005; revised 14 December 2005; accepted 14 December 2005

Available online 7 February 2006

Among the various phenotypes seen in Down syndrome (DS), mental

retardation is the most common and most debilitating condition

suffered by individuals with DS. The DYRK1A gene on human

chromosome 21q22.2 encodes a subfamily of protein kinases that

displays dual substrate specificities and is known to play a critical

role in neurodevelopment. To study DS mental retardation, we have

generated transgenic mice that contain only one copy of the

complete human DYRK1A gene in a bacterial artificial chromosome.

The transgenic mice showed significant impairment in hippocampal-

dependent memory tasks in a Morris water maze. Interestingly, we

observed shifts in both long-term potentiation and long-term

depression, which suggests a role for DYRK1A in bidirectional

synaptic plasticity. These mice represent the most clinically relevant

DYRK1A mouse model to date and provide us a valuable tool for

the in vivo study of mechanisms that underlie the learning and

memory deficit in DS.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Down syndrome; DYRK1A; Mental retardation; Learning and

memory; Transgenic mice; Bacterial artificial chromosome

Introduction

Down syndrome (DS) is the most common genetic disorder,

with a frequency of 1 in every 700 to 800 live births, and is caused

by the presence of an extra copy of all or part of human

chromosome 21 (Jacobs et al., 1959; Lejeune et al., 1959). In

addition to a characteristic set of physical features, DS individuals

0969-9961/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.nbd.2005.12.006

* Corresponding authors. Fax: +82 42 865 6690.

E-mail addresses: ilhoha@inje.ac.kr (I. Ha), wjsong@inje.ac.kr

(W.-J. Song).1 The authors wish it to be known that, in their opinion, the first two

authors should be regarded as joint First Authors.

Available online on ScienceDirect (www.sciencedirect.com).

show a variety of other anomalies, including low muscle tone,

congenital heart defects, gastrointestinal malformations, immune

and endocrine system defects, a high incidence of leukemia, and

early onset of dementia of the Alzheimer type. Individuals with DS

also exhibit mild to severe mental retardation, which is a major

factor in preventing these patients from leading fully independent

lives (Korenberg et al., 1994; Patterson, 1987; Pulsifer, 1996). As

is the case with DS individuals, the Ts65Dn mouse, a typical

murine model for DS, is segmentally trisomic for a region of the

mouse genome (APP to MX1) that is homologous to part of the

long arm of human chromosome 21 and displays behavioral and

learning defects as well as neurodegeneration of basal forebrain

cholinergic neurons (Demas et al., 1998; Holtzman et al., 1996;

Reeves et al., 1995).

Through efforts to isolate the gene(s) responsible for DS mental

retardation, a candidate gene, DYRK1A, was identified (Guimera et

al., 1996; Kentrup et al., 1996; Shindoh et al., 1996; Song et al.,

1996). DYRK1A is a mammalian orthologue of the Drosophila

minibrain (mnb) gene which is essential for normal postembryonic

neurogenesis (Tejedor et al., 1995). The DYRK1A gene is localized

on the human chromosome 21q22.2 region, which has been

associated with a variety of anomalies, including mental retardation

(Korenberg et al., 1990; Rahmani et al., 1989; Sago et al., 2000).

The human (DYRK1A) and rodent (Dyrk1A) genes are ubiquitously

expressed in fetal and adult tissues, with strong expression in the

brain and heart (Guimera et al., 1996; Hammerle et al., 2003a,b;

Marti et al., 2003; Rahmani et al., 1998; Song et al., 1996). Both

the DYRK1A mRNA in DS fetal brains and the corresponding

mouse Dyrk1A mRNA in Ts65Dn mice have been shown to be

overexpressed (Guimera et al., 1999).

DYRK1A is a member of the DYRK [Dual-specificity

Tyrosine(Y) Regulated Kinase] family, which contains seven

mammalian members (DYRK1A, DYRK1B, DYRK1C; DYRK2;

DYRK3; and DYRK4A, DYRK4B) (Becker and Joost, 1999). As

its name implies, the DYRK1A enzyme has dual substrate

K.-J. Ahn et al. / Neurobiology of Disease 22 (2006) 463–472464

specificities; autophosphorylation for self-activation takes place on

the tyrosine-321 residue in the active loop of the catalytic domain

(Kentrup et al., 1996; Lochhead et al., 2005), and target protein

phosphorylation occurs on serine/threonine residues. DYRK1A

phosphorylates or interacts with several proteins, including STAT3,

FHKR, Gli-1, eIF2B(, Tau, dynamin, glycogen synthase, 14-3-3,

CREB, cyclin L2, Arip4, Hip-1, and PAHX-AP1. Its diverse array

of interactions suggests that DYRK1A participates in multiple

biological pathways (Galceran et al., 2003; Hammerle et al.,

2003a,b). The first function hypothesized for DYRK1A was a role

in DS mental retardation and was deduced from the function of its

Drosophila orthologue, the minibrain (mnb) gene. mnb encodes a

serine/threonine protein kinase that is required for normal

postembryonic neurogenesis. Flies that carry mutations in the

mnb gene express at 30–60% mnb protein level compared to that

of wild-type and display a specific and marked size reduction in the

optic lobes and central brain hemispheres as well as behavioral

deficiencies (Tejedor et al., 1995). In the worm Caenorhabditis

elegans, the addition to the genome of extra copies of its DYRK1A

homologue, mbk-1 , causes behavioral defects in chemotaxis

toward volatile chemoattractants (Raich et al., 2003).

The critical role of DYRK1A in DS mental retardation was

further strengthened by analyzing a variety of genetically altered

mice. Dyrk1A knockout (KO) mice show a general growth delay

and are embryonic lethal (E14.5), which strengthens the notion that

Dyrk1A has myriad vital and non-redundant biological functions

(Fotaki et al., 2002). Mice that are heterozygous for Dyrk1A

(Dyrk1A +/�) show decreased neonatal viability, and their brain

and body sizes are smaller than those of control littermates.

Transgenic mice that carry a yeast artificial chromosome (YAC)

that bears human DYRK1A genomic DNA (the DYRK1A YAC

mice) show learning defects (Smith et al., 1997). And, finally,

transgenic mice that carry extra copies of the rodent Dyrk1A cDNA

(the Dyrk1A cDNA mice) exhibit neurodevelopmental delay as

well as motor abnormalities and cognitive deficits similar to those

seen in DS individuals (Altafaj et al., 2001; Martinez de Lagran et

al., 2004). Although these various genetically altered mice have

provided researchers with valuable information, each possesses

pitfalls when used to investigate the function of DYRK1A. The

inclusion of additional human genes (e.g., TTC3, DSCR3) in the

YAC clone and the use of a heterologous promoter (the inducible

sMT-Ia promoter) to drive the expression of the rodent Dyrk1A

cDNA could lessen the experimental results obtained with these

animal models.

In this study, we generated DYRK1A transgenic mice using a

bacterial artificial chromosome (BAC) clone that contained only

the complete DYRK1A genomic DNA fragment, which included

the endogenous human promoter. Analyses of these DYRK1A

BAC transgenic (BAC TG) mice showed a single chromosomal

integration of the DYRK1A BAC DNA and about a 1.5-fold

overexpression of Dyrk1A (mouse Dyrk1A plus human DYRK1A)

protein compared to non-transgenic control littermates. Therefore,

the presence of the third copy of the DYRK1A gene and the

increased mRNA expression seen in DS fetal brain were mimicked

in the BAC TG mice. The BAC TG mice showed severe learning

and memory deficiencies in a Morris water maze test, and

electrophysiological analyses revealed alterations in two forms of

synaptic plasticity, long-term potentiation (LTP; increased) and

long-term depression (LTD; decreased), resulting in so-called

bidirectional synaptic plasticity changes. Taken together, our

findings show that BAC TG mice constitute a clinically relevant

animal model for the study of mechanisms that underlie the

learning and memory deficit resulting from overexpression of

DYRK1A.

Materials and methods

Animals

Mice were housed in cages (in groups of 3 or 4 mice) with food

and water freely available and under a standard specific pathogen-

free (SPF) vivarium. Humidity (50 T 10%) and temperature (22 T2-C) were kept constant, and a 12-h light/dark cycle was used

(lights on, 7:00 a.m. to 7:00 p.m.). All tests were conducted during

the light cycle, and the procedures were performed in accordance

with guidelines set forth by the Inje University Council Directive

for the proper care and use of laboratory animals.

Production of human DYRK1A BAC transgenic mice

Standard microinjection procedures were used for transgenic

mice production (Macrogen, Seoul, Korea). Briefly, fertilized

mouse eggs were flushed from the oviducts of superovulated

C57BL/6NCrjBgi mice, and male pronuclei were injected with

777J19 BAC DNA (2 ng/Al) that had been linearized with the PI

SceI enzyme at its cognate restriction site located in the

pBACe3.6 vector. The injected eggs were reimplanted in the

oviducts of pseudo-pregnant ICR recipient females. At 3 weeks

of age, the animals were tested for the presence of the transgene

by PCR analysis of their genomic DNA using the following three

primer pairs: 1. T7 (5V-TAATACGACTCACTATAGGG) and

777J19 T7 (5V-ATAATTTCATAAATTTTCCCAG) for the T7 side.2. SP6 (5V-ATTTAGGTGACACTATAG) and 777J19 SP6 (5V-TTAAACTGGTCCAGGTCTGG) for the SP6 side. 3. internal-F

(5V-GGAGCAGTTACTTTACTTAAATC) and internal-R (5V-CACAACACAAAACAATACAACTG) for the internal sequence.

PCR conditions were as follows: initial incubation, 94-C for 5 min

(one time); cycle conditions, 94-C for 30 s, annealing temperature

for 30 s, and 72-C for 30 s (35 cycles); one final elongation for 1 min

at 72-C (one time). The annealing temperatures were 52-C for the T7

and SP6 side and 57-C for the internal sequence.

FISH

To prepare slides of mouse cells in interphase, peripheral blood

sample (100 Al) that had been drawn from the veins of each mouse

was treated with a 75 mM KCl hypotonic solution and then fixed in

a methanol:acetic acid (3:1) solution. The biotinylated probe was

made using a biotin-nick translation kit (Roche, Basel, Switzer-

land) with 500 ng of 777J19 BAC clone DNA. The treated blood

sample was placed on a slide and then hybridized overnight at

37-C with 30 ng of the biotinylated probe along with 5 Ag of

mouse Cot-1 DNA, 10 Ag of herring sperm DNA, and 1 Ag of yeasttRNA. The next day, the sample on the slide was blocked by

incubation with TNT buffer (100 mM Tris–HCl, pH 8.0, 150 mM

NaCl, 0.05% Tween 20) containing 0.5% blocking reagent for 45

min at room temperature in a humidified box. The blocking step

was followed by subsequent incubation of the sample with

fluorescein avidin DCS, biotinylated goat anti-avidin D, and

amplification of the signal was achieved by subjecting the sample

to a second incubation with fluorescein avidin DCS (Vector Lab.,

K.-J. Ahn et al. / Neurobiology of Disease 22 (2006) 463–472 465

Burlingame, CA, USA). The slide sample was then counterstained

with propidium iodide (final concentration, 1 Ag/ml in Vectashield,

Vector Lab.), and the signal was detected using fluorescence

microscopy.

Quantitative PCR

Following primers and conditions were used for quantitative

PCR to determine the copy number of the integrated BAC DNA: for

the detection of mouse specific transcript, MF (5V-AGGTGCGCCAGCAGTTTCCG-3V) and MR (5V-ATGCAATGCGTTCT-GCTGG-3V); for the detection of mouse and human transcripts,

MHF (5V-CTATGGAGGTTGGCCACAGT-3V) and MHR (5V-GGGATTGGAGTAGACGGTC-3V). Real-time PCR was carried

out using LightCycler FastStart DNAMaster SYBRGreen I (Roche,

Basel, Switzerland) and LightCycler (Roche) as recommended by

the manufacturers. The condition of real-time PCR was 95-C min

for 10 min, 25 cycles of 95-C for 10 s, 60-C for 5 s, and 72-Cfor 10 s.

DYRK1A antibody

An anti-DYRK1A antibody was raised by immunizing rabbits

with the C-terminal DYRK1A peptide as previously described

(Okui et al., 1999). The DYRK1A-specific antibody was further

purified from the serum on an immunogen-peptide coupled Affi-

gel 10 affinity column (Bio-Rad, Hercules, CA, USA) by

subjecting the bound antibody to acidic (0.1 M glycine, pH 2.5)

and basic (0.1 M triethylamine, pH 11.5) treatment. The purified

antibody recognized both the human DYRK1A and mouse

Dyrk1A proteins.

Immunohistochemistry

Mice were anesthetized with isoflurane and perfused trans-

cardially with phosphate-buffered saline (PBS, pH 7.4) followed by

fixative (4% paraformaldehyde in PBS, pH 7.4). The brains were

fixed at 4-C overnight and were cryoprotected. Sagittal sections (16

Am thick) were incubated with anti-DYRK1A antibody (a 1:200

dilution) and biotinylated anti-rabbit IgG (a 1:200 dilution) followed

by treatment with avidin biotinylated horseradish peroxidase (a 1:50

dilution, Vector Lab.). After visualization with the SG reagent set

(Vector Lab.), sections were examined with a Zeiss Axioplan

microscope using conventional optics.

Behavioral test

Morris water maze tests were performed in an SPF facility with

two independent transgenic mice (TG lines 36 and 46) that were

established by crossing with C57BL/6NCrjBgi mice. Mice were

trained in a 1.5 m diameter water maze under the following

conditions: opaque water at 24 to 27-C; an escape platform (12 cm

in diameter) placed 0.5 cm below the water surface; a 10 min inter-

trial interval; maximum trial duration 60 s with 20 s on the

platform at the end of each trial. Twelve control littermates and 11

BAC TG mice in each line received four trials per day for 8 days.

To test whether the mice remembered the location of the platform,

probe trials (30 s in duration after removal of the platform) were

performed 24 h after the fourth and sixth training sessions and 5

days after completion of the hidden platform training protocol. The

cue tests were performed to test the swimming speed and visual

ability using the black-visible platform at 5 days after the last

training session. All the trials were recorded and traced with an

HVS Image tracking system (Hampton, UK).

Electrophysiology

Freshly dissected hippocampal slices (400 Am thick) were

maintained in a storage chamber containing artificial cerebrospinal

fluid (ACSF; 124 mM NaCl, 5 mM KCl, 1.25 mM NaH2PO4, 3

mM MgCl2, 1 mM CaCl2, 26 mM NaHCO3, and 10 mM dextrose,

pH 7.4) at 30-C as described (Saura et al., 2004). Stimulation (200

As) pulses were delivered with a bipolar concentric metal electrode.

Synaptic strength was quantified as the initial slope of field

potentials recorded with ACSF-filled microelectrodes (1 to 2 MV).

Baseline responses were collected at 0.07 Hz with a stimulation

intensity that yielded a half-maximal response. LTP was induced

by five episodes of theta-burst stimulation (TBS) delivered at 0.1

Hz. Each episode contained ten stimulus trains (4 pulses at 100 Hz)

delivered at 5 Hz. Average responses (mean T SEM) are expressed

as percent of pre-TBS baseline response. LTD was induced with

900 paired-pulses (40 ms apart) delivered at 1 Hz. Repeated

measures ANOVA and non-paired t tests were used to assess the

statistical significance of the results.

Results

Production of DYRK1A BAC TG mice

To produce a mouse model that displays the mental retardation

observed in patients with DS, the following criteria should be

satisfied: (i) temporal and spatial DYRK1A expression in the

animal model should mimic that of the endogenous gene; (ii) the

amount of DYRK1A overexpression in the animal should be similar

to that observed in DS brains; and (iii) the animal should exhibit

learning and memory deficits similar to those seen in DS patients.

Because cDNA that has been fused to an exogenous promoter

might display non-endogenous expression patterns, we used

DYRK1A genomic DNA that contained the endogenous promoter

for transgenic mice production. We selected the BAC vector as a

vehicle for the genomic DYRK1A DNA as this vector can carry an

insert of up to 200 kb, and the DYRK1A genomic DNA fragment

that we used was about 170 kb. Furthermore, BAC clones are

easier to manipulate and less likely to contain additional genes than

are YAC clones, thus allowing the study of a single-gene effect.

We searched the Human BAC End database (http://www.

tigr.org/tdb/humgen/bac_end_search/bac_end_intro.html) to iden-

tify BAC clones that contained only the human DYRK1A genomic

fragment. One clone from the RPCI-11 human BAC library,

777J19, contains the full-length 168.5-kb human DYRK1A

genomic DNA fragment, including all 14 exons (Guimera et al.,

1999; Hattori et al., 2000). The 777J19 clone also contains an 18.5-

kb stretch of DNA upstream of exon 1 and a 3-kb untranslated 3Vend sequence downstream of the last exon (Fig. 1A). Analyses of

the 5V and 3V DNA sequences revealed that the 777J19 clone does

not contain any additional genes (the 5V and 3V neighboring genes

are at least 110 kb away) and includes the DYRK1A promoter and a

poly-A tail consensus sequence. Therefore, we concluded that the

777J19 BAC clone contains only the DYRK1A gene in its entirety.

Among three founder mice obtained from the microinjection of

fertilized eggs, two mouse lines (BAC TG lines #36 and #46) were

Fig. 1. Production of DYRK1A BAC TG. (A) Schematic map of the

777J19 BAC clone used for DYRK1A BAC TG mice production. PI SceI

is a unique restriction enzyme used to linearize the BAC clone for

microinjection. Open box, vector pBACe3.6; filled boxes represent 14

exons of DYRK1A. 3V-UTR, 3V untranslated region. (B) Interphase FISH

of control and BAC TG mice. Arrows show the BAC DNA probe-

hybridized signals.

K.-J. Ahn et al. / Neurobiology of Disease 22 (2006) 463–472466

used in this study. Chromosomal integration of the complete BAC

clone was confirmed by checking for the presence of the flanking

ends (the T7 and SP6 sides in the pBACe3.6 vector) and by

assessing the integrity of the internal sequences by polymerase

chain reaction (PCR) (data not shown). To determine the number of

the chromosomal integration, fluorescent in situ hybridization

(FISH) was employed. Under the hybridization conditions we

used, the human BAC probe hybridized with the integrated

DYRK1A BAC DNA, but not with the mouse Dyrk1A gene. FISH

experiments performed with the human BAC probe and cells from

the BAC TG mice revealed that the interphase portion of the cell

division cycle showed a single spot in each nucleus for both mouse

lines, indicating the integration into a single chromosome site;

interphase cells from non-transgenic (‘‘control’’) littermates

showed no detectable signal in FISH experiments (Fig. 1B). The

birth ratio of BAC TG mice to control littermates was close to

1:1, also suggesting that a BAC DNA had integrated into a single

chromosome. The integrated BAC DNA copy number was

measured by quantitative PCR with genomic DNA, using

mouse-specific Dyrk1A primers for normalization and primers

for both human DYRK1A and mouse Dyrk1A for detection for

total copy number. The ratio of the human + mouse copy number

to mouse copy number for the BAC TG mouse was close to 1.5,

indicating that one copy of the BAC clone was integrated into the

genome, a finding that supported the FISH results (data not

shown).

To compare the total DYRK1A expression levels in BAC TG

mice and control littermate mice, we prepared total and fraction-

ated brain lysates from these animals. When we subjected the same

amount (75 Ag of protein) of the BAC TG and control mice whole-

brain lysates to Western blotting, the Dyrk1AT band intensity

(which includes signals from both human DYRK1A and mouse

Dyrk1A) in the BAC TG mice lysate was stronger than that of the

control mice lysate, while h-actin band intensities were similar in

both lysates (Fig. 2A). The intensity of the Dyrk1AT band obtained

when 50 Ag of protein from the BAC TG brain lysates was

subjected to Western blotting was equivalent to that obtained when

75 Ag of protein from the control brain lysates was treated in the

same manner. This finding indicates that there is an ¨1.5-fold

overexpression of the Dyrk1AT protein in the BAC TG mice. This

¨1.5-fold overexpression of Dyrk1AT in BAC TG mice agrees

well with the reported level of overexpression of DYRK1A mRNA

in DS brains and Dyrk1A mRNA in Ts65Dn mice brain (Guimera

et al., 1999). Quantification of Dyrk1AT protein expression in the

BAC TG whole-brain lysate showed an average of 1.5-fold

overexpression for both BAC TG mouse lines (Fig. 2B). Dyrk1A

expression in specific brain regions was also compared. While the

expected ¨1.5-fold overexpression of Dyrk1AT protein in BAC

TG mice in prefrontal cortex was observed, higher overexpression

(¨2.5-fold) was detected in the hippocampus of the BAC TG mice

(Fig. 2C). Finally, the Dyrk1AT overexpression of the BAC TG

mice in the cortex and cerebellum showed a similar ¨1.5 times that

in control mice (data not shown). The Dyrk1AT expression patterns

in the hippocampus, where electrophysiological experiments were

performed, were also analyzed by immunohistochemistry (Fig.

2D). Dyrk1A immunostaining of control mouse hippocampus

showed an expression pattern that was similar to previously

reported results (Marti et al., 2003): weak Dyrk1A expression was

detected in the pyramidal cell layer of the hippocampus and the

granular cell layer of the dentate gyrus. In contrast, Dyrk1AT

expression in BAC TG mice was increased in these brain regions,

especially in the granular cell layer of the dentate gyrus. We

analyzed the brain weights of BAC TG and control mice. At 9

weeks, the body weights (19.6 T 0.5 g, n = 9) of BAC TG mice

were not different from those of control mice (19.6 T 0.3 g, n = 6).

However, BAC TG mice showed a 19% increase in brain weights

(533 T 6 mg) when compared to those of control mice (448 T 12

mg, P < 0.001).

Defect of spatial memory in BAC TG mice

To test hippocampal-dependent spatial cognition, two BAC TG

mouse lines were trained in the standard Morris water maze with a

hidden platform (Morris et al., 1982). The training consisted of

eight sessions (4 trials/session, 10-min inter-trial intervals). The

ability of control mice to escape to the hidden platform improved

during the training trials, as evidenced by a reduced searching error

(Fig. 3A). However, the BAC TG mice group did not show any

improvement in finding the hidden platform, and these results were

significantly different from those of control mice group (P < 0.05

or 0.01, repeated ANOVA). Even after repeated training for 8 days,

the searching errors of BAC TG mice were not decreased,

indicating severe memory impairments. To test whether the mice

had indeed learned the spatial location of the hidden platform, we

conducted probe trials in which the platform was removed after the

fourth, sixth, and eighth days of training. For the control group, the

number of crossings of the annulus of the hidden escape platform

Fig. 2. Overexpression of DYRK1A protein in BAC TG. (A) DYRK1AWestern blotting of mouse whole-brain lysates from 4-week-old control and BAC TG

(TG line #36) mice. Proteins were separated by electrophoresis on an 8% SDS polyacrylamide gel and were subjected to Western blotting analysis with an anti-

DYRK1A antibody. h-actin bands were used for normalization. (B) Quantification of DYRK1A overexpression in the whole-brain lysate. Data are shown as

mean T SEM (n = 5). **P < 0.01. (C) DYRK1A expression in hippocampus and prefrontal cortex (BAC TG: line #36). *P < 0.05; **P < 0.01. (D) DYRK1A

immunohistochemistry of the sagittal sectioned mouse hippocampus from control and BAC TG mice. DG, dentate gyrus.

K.-J. Ahn et al. / Neurobiology of Disease 22 (2006) 463–472 467

was increased up to 2 times during the 2nd and 3rd 30-s probe

trials when compared with the 1st trial. In contrast, the BAC TG

mice did not show any improvement with training (P < 0.05,

repeated ANOVA, Fig. 3B). These observations support the

conclusion that the BAC TG mice did not learn the location of

the hidden platform. In cue tests, where the goal was to find visible

platforms in the water maze, the performance of the BAC TG mice

and control littermates did not differ, demonstrating that the

learning impairments displayed by the BAC TG mice did not

result from differences in swimming speed or visual ability (data

not shown). Moreover, although the BAC TG mice showed a

slight delay in balancing during rotarod training at the first trial

(fixed speed at 16 rpm; Student’s t test, not significant), they

maintained their balance in subsequent training sessions (fixed

speeds, 24 and 32 rpm, and variable speeds; maximum duration

100 s), as did their control littermates (Fig. 4). Overall, we did not

detect any apparent motor abnormalities in BAC TG mice, which

would affect the interpretation of the Morris water maze test

results. Both BAC TG mouse lines showed similar defects in

spatial memory, confirming that the observed phenotypes did not

result from non-specific effects of BAC DNA integration into the

chromosome.

Bidirectional synaptic plasticity in BAC TG mice

Neuronal processing occurs when chemical signals (neuro-

transmitters) from the presynaptic cell give rise to short- or long-

term changes in the membrane potential of the postsynaptic cell.

This signal transduction is crucial for neurotransmission and

synaptic plasticity. The memory deficits observed in the water

maze tests suggested that BAC TG mice might have changes in

such synaptic functions. To determine whether there was any

change in synaptic plasticity in the BAC TG mice, we performed a

series of electrophysiological recordings on the Schaffer collateral-

CA1 synapse of hippocampal slices. We first measured the initial

slope of the evoked field excitatory postsynaptic potential (fEPSP)

and the amplitude of the fiber volley (FV), which is a measure of

the number of recruited axons. These measurements allow one to

assess basal synaptic transmissions and did not differ in BAC TG

and control mice, indicating the presence of normal basal synaptic

transmission in the BAC TG mice (data not shown). To measure

synaptic plasticity in BAC TG mice, we assessed LTP and LTD in

these animals. Administration of theta-burst stimulation (TBS) at

100 Hz yielded an increase in LTP in BAC TG mice (174 T 12%)

that was higher than that in the control mice (139 T 11%, P < 0.05,

ANOVA for response at 5 min; Fig. 5A). The magnitude of the

LTP measured for 60 min after TBS in BAC TG mice (173 T 13%)

also was significantly increased relative to that of the control mice

(146 T 11%; F[63,3969] = 1.35, P < 0.05). In addition, LTD at

paired-pulse low frequency stimulation (900 pulses at 1 Hz) was

significantly reduced in the BAC TG hippocampal slices relative to

the control (Fig. 5B). Furthermore, in BAC TG mice, the

magnitude of the LTD measured for 65 min after conditioning

(90.2 T 5.1%) was higher than that of the control mice (79.0 T4.5%; F[53,2703] = 2.03, P < 0.001). Finally, stimulation at an

intermediate frequency (900 pulses at 10 Hz) produced a

modestly enhanced potentiation in the BAC TG mice relative

to control mice (F[55,1485] = 1.35, P < 0.05; Fig. 5C). Taken

together, these results show that DYRK1A overexpression

caused altered bidirectional synaptic plasticity, providing a cel-

Fig. 3. Learning and memory defects of DYRK1A BAC TG mice. (A)

Searching error for BAC TG mice (TG line #36, closed circles) and control

littermates (open circles). Two-month-old mice were used for the Morris

water maze test [11 BAC TG mice (6 female and 5 male) and 12 control

littermates (6 female and 6 male)]. Searching error was calculated by

summing up the distance (in meter) between a mouse and the platform

every second. (B) Probe test. After removal of the hidden platform,

crossings of the annulus of the platform were measured after the fourth (first

probe trial), sixth (second probe trial), and eighth (third probe trial) training

trials. Data are shown as mean T SEM (n = 12 for control, n = 11 for BAC

TG). *P < 0.05; **P < 0.01.

K.-J. Ahn et al. / Neurobiology of Disease 22 (2006) 463–472468

lular basis for the memory deficits observed in the BAC TG

mice (Fig. 5D).

Fig. 4. Normal motor activity of BAC TG mice. (A) Fixed speeds. (B)

Variable speeds. Rotarod tests were performed for a total of 6 different

speeds with a five-station treadmill (ENV-575M, Med. Associate Inc., St.

Albans, VT, USA). We first used a lower fixed speed (16 rpm), then

progressed to a higher speed (32 rpm), and then to variable speeds. The

same mice used for the Morris water maze test were used for the rotarod

test. The maximum duration time was 100 s. Data are shown as mean T

SEM (n = 12 for control, n = 11 for BAC TG).

Discussion

Several genes (SOD1, SIM2, S100h, and DYRK1A) localized

on chromosome 21 are known for their involvement in the normal

function of learning and memory, thus contributing to the mental

retardation phenotype of DS when overexpressed (Ema et al.,

1999; Harris-Cerruti et al., 2004; Smith et al., 1997; Winocur et al.,

2001). Transgenic mice of those genes show mild to severe

impairment in the learning and memory ability. Among them,

DYRK1A is the most studied gene responsible for the learning and

memory deficit seen in DS. However, we still know little about the

molecular mechanism(s) that underlie(s) the learning and memory

deficit resulting from overexpression of DYRK1A. Because

DYRK1A phosphorylates or otherwise interacts with a variety of

proteins, including transcription factors, one would predict that

DYRK1A participates in multiple biological pathways (Galceran et

al., 2003). However, it is not known which, if any, of these

interacting proteins or their biological pathways are involved in

generating the learning and memory deficits observed in the BAC

TG mice or in DS patients.

One of the aims of this study was to produce a clinically

relevant DYRK1A mouse model in which to study DS mental

retardation. To this end, we produced a TG mouse that carries one

copy of the human DYRK1A gene on a BAC. We do not know

whether the 18.5-kb promoter region upstream of the first exon of

the BAC-borne DYRK1A gene contains sufficient promoter

information for proper regulated expression of DYRK1A in the

BAC TG mice. However, the fact that genomic integration of a

single BAC clone in the BAC TG mice resulted in an ¨50%

increase in expression of the DYRK1A protein, compared to

control mice, suggests that the functional transcriptional regulatory

elements are present in the 18.5-kb region. One possible experiment

to test if the 18.5-kb region contains the necessary regulatory

elements is to breed BAC TG mice with the heterozygous Dyrk1A

(+/�) TG mice that show decreased neonatal viability, develop-

mental delay, and smaller brain and body sizes. If the 18.5-kb region

contains the necessary elements, the heterozygous Dyrk1A (+/�)

Fig. 5. Bidirectional synaptic plasticity in the hippocampal Schaffer collateral-CA1 synapse of BAC TG mice. (A–C) Extracellular recordings in the CA3–

CA1 pathway from hippocampal slices of BAC TG mice (filled circles) and control mice (open circles). (A) Hippocampal LTP induced by theta-burst

stimulation (TBS) was enhanced in BAC TG mice relative to control mice. (B) Hippocampal LTD induced by paired-pulse LFS was reduced in BAC TG mice

relative to control mice. (C) Stimulation at 10 Hz produced an enhanced potentiation in BAC TG mice relative to control non-TG mice. (D) Summary of fEPSP

changes from traces of the final 5 min with different stimulation frequencies. (n = 10, 34), for example, indicates that 34 slices from 10 mice were used for

recordings.

K.-J. Ahn et al. / Neurobiology of Disease 22 (2006) 463–472 469

TG mice carrying the DYRK1A BAC DNAwill have all or part of

the restored phenotypes, depending on the similarity of the

promoters between human and mouse.

BAC TG mice showed a 19% heavier brain weights than those

of control mice. This observation is comparable with the results,

17% and 14% increase in brain weight, from two YAC TG mouse

lines which used the endogenous DYRK1A promoter for DYRK1A

expression (Branchi et al., 2004). We did not observe any

detectable difference in body weight or other organs (spleen, liver,

and heart), suggesting the brain-specific role of the DYRK1A gene.

The BAC TG mice have several advantages for the study of the

learning and memory deficit in DS compared to previous DYRK1A

mouse models (Altafaj et al., 2001; Smith et al., 1997). First, the

BAC TG mice contain only an extra copy of the DYRK1A genomic

DNA, which allows us to study, in isolation, the effects of

overexpression of DYRK1A in vivo. Second, while multiple

genomic integration sites for the Dyrk1A cDNA clones were

detected in the corresponding TG mice, the BAC TG mice

contained only one copy of the DYRK1A gene integrated into its

genome. This fact makes our model more clinically relevant than

previous models as DS phenotypes result from an extra copy of

chromosome 21 (and, thus, of DYRK1A). Third, unlike the cDNA-

containing TG mice, which make use of an exogenous promoter,

the expression of DYRK1A in the BAC TG mice is tightly

controlled by the endogenous promoter to drive DYRK1A

expression. Fourth, the BAC TG mice showed severe spatial

memory defects. The severity of the learning deficits reported for

the DYRK1A YAC and cDNA TG mice was milder than that of

Ts65Dn (Demas et al., 1998; Escorihuela et al., 1998). These

various differences could result from the divergent genetic back-

grounds of the mice, the ages of the tested mice, experimental

environments, the nature of the clones used for transgenic mouse

production, or the existence of additional genes integrated in the

genome. Hypoactivity in YAC TG (Smith et al., 1997) and motor

abnormalities in cDNA TG (Altafaj et al., 2001; Martinez de

Lagran et al., 2004) mice were reported. In contrast, we did not

observe significant motor abnormalities for two different BAC TG

mouse lines. For example, in a Morris water maze test, the

swimming speeds recorded for the BAC TG mice did not differ

from those of control littermates. Furthermore, like control

littermates, BAC TG mice were able to learn quickly how to

maintain their balance on the rotarod even at the highest speed

(variable speed, 4–40 rpm). The presence of motor abnormalities

in the other Dyrk1A mouse models might be explained by

differences in testing time (short vs. extended), the age of each

mouse tested (young vs. old), or the mouse strains used for

transgenic mice production (Crabbe et al., 1999).

We found that the significant spatial learning and memory

defects of the BAC TG mice were associated with altered

hippocampal LTP and LTD. Indeed, we detected a shift in

plasticity toward a greater induction of LTP and a lesser induction

of LTD. Synaptic plasticity in the hippocampus is known to be

K.-J. Ahn et al. / Neurobiology of Disease 22 (2006) 463–472470

mediated by the activation of AMPA (a-amino-3-hydroxy-5-

methyl-4-isoxazole propionate) and NMDA (N-methyl-d-aspar-

tate) receptors, an increase in spinal Ca2+ concentrations, and

subsequent activation of protein kinases and/or phosphatases

modulated by the spinal Ca2+ concentrations (Lisman, 2001;

Paulsen and Sejnowski, 2000). The activation of NMDA receptors

and the nature of the increase in postsynaptic spinal Ca2+

concentrations are key factors that determine whether LTP or

LTD will be induced. If a certain synaptic factor or event is altered,

the plasticity pattern for a given mode of stimulation would be

changed. For example, an altered synaptic factor might make the

synapse prefer only one type of plasticity because the mutated

factor now easily induces LTP, while it barely affects LTD (or vice

versa). Such a change might bring about the bidirectional changes

in synaptic plasticity seen in the BAC TG mice.

According to mathematical analyses, bidirectional changes in

synaptic strength affect behavior more significantly than do

unidirectional synaptic changes (Morris and Willshaw, 1989).

Our results clearly show that the shifted bidirectional plasticity.

The following question is why and how the overexpression of

DYRK1A changes the synaptic plasticity in that manner. Based on

the previous examples showing the shifted bidirectional plasticity,

one of the possible mechanisms is that DYRK1A acts on the

NMDA/AMPA receptor activity and/or Ca2+-dependent protein

phosphorylation. One of the examples is the PSD95 knockout

mice. PSD95 is an anchoring protein that affects NMDA receptor

signaling, and PSD95 KO mice show a plasticity preference that is

shifted toward LTP (Migaud et al., 1998). It means that the

crosstalk between NMDA receptors and their binding partners is

important to keep the synaptic plasticity to the normal level. One

group of the binding partners is the postsynaptic protein kinases

including Ca2+/calmoduline-dependent kinase, and protein kinase

C. In our hypothesis, the protein kinase activity of DYRK1A is

suggested to contribute the phosphorylation of postsynaptic

proteins as other protein kinases move the bidirectional plasticity

toward LTP side. Another evidence supporting this hypothesis

comes from the previous study reporting that the plasticity

preference is also shifted toward LTP in KO mouse models that

lack either the gene encoding calcineurin (a Ca2+-dependent

protein phosphatase involved in signaling) (Zeng et al., 2001).

Actually, the protein kinase and the protein phosphatase make the

balance of phosphorylation and dephosphorylation of the synaptic

proteins which induce LTP and LTD subsequently. Thus, the lack

of calcineurin (which means the lack of phosphorylase activity)

shifts the balance of the phosphorylation state of synaptic proteins

and makes more LTP. In this hypothesis, the increased phosphor-

ylation power in synapse of DYRK1A overexpressing mice would

result in the same manner of the changes in synaptic plasticity.

It is not yet clear that DYRK1A does really act on the

phosphorylation of the postsynaptic proteins in the dendritic spine.

However, the kinase activity is not always required in the

phosphorylation state of synaptic protein. The other previous case

showing the shifted bidirectional plasticity comes from the gene

encoding the p21-activated protein kinase (Hayashi et al., 2004).

The deficiency of the p21-activated protein kinase yields an

enlarged spine and an increased amount of AMPA receptor in

postsynaptic membranes, relative to a control. Although it is not

clear that the p21-activated protein kinase acts on the phosphor-

ylation state of the postsynaptic proteins, its protein kinase activity

modulates the cytoskeleton and spine structure so as to modify the

NMDA/AMPA receptor signaling. If reasoning by analogy, it is

possible for DYRK1A to modulate not the synaptic protein but the

cytoskeletal structure of synaptic spines. It remains to be

investigated the target mechanism of DYRK1A. At least, it is

provocative that each of these proteins is related to NMDA/AMPA

receptor activity and/or Ca2+-dependent protein phosphorylation.

Because DYRK1A shows a similar manner of modulation of

plasticity with the previous examples showing shifted bidirectional

plasticity mentioned above, it is possible that DYRK1A has a role

in synaptic spine structure and/or glutamate receptor signaling.

The direction of the hippocampal synaptic plasticity of BAC

TG mice appears to be opposite to those of other DS model mice.

In Ts65Dn mice, which are segmentally trisomic for a region

(spanning from APP to MX1) that is homologous to human

chromosome 21, LTD is increased and LTP is decreased (Costa and

Grybko, 2005; Kleschevnikov et al., 2004; Siarey et al., 1999).

Another segmental trisomic mice (SOD1-MX1), Ts1Cje, also

showed the similar changes in LTD and LTP (Siarey et al.,

2005). However, the shifted direction of synaptic plasticity seen in

BAC TG mice is not necessarily contradictory to that of Ts65Dn or

Ts1Cje mice which carry the other extra genes in addition to

Dyrk1A. The possible explanation is that the decreased LTP and

increased LTD of other models result from the presence of the

additional genes while overexpressed Dyrk1A may partially

contribute to the shift of the synaptic plasticity to the opposite

direction. The synaptic plasticity shift of BAC TG mice was not as

much as that of Ts65Dn or Ts1Cje mice to overcome the marked

shift. A way to address this hypothesis is to cross a Ts65Dn or

Ts1Cje mouse with a heterozygous Dyrk1A (+/�) mouse to

produce a trisomic mouse with only two copies of Dyrk1A gene

and to test for the further shift in synaptic plasticity. A few proteins

expressed from chromosome 21 are known to be involved in the

altered LTP and/or LTD. Calcium-binding protein S100h knockout

mice showed the strengthened synaptic plasticity (Nishiyama et al.,

2002), and copper/zinc superoxide dismutase SOD1 transgenic

mice have an impaired LTP (Gahtan et al., 1998). It will be

interesting to see if the altered synaptic plasticity by those proteins

could be recovered, at least partially, by DYRK1A overexpression

in double transgenic mice (S100h/DYRK1A, SOD1/DYRK1A).However, further investigation should be carried out to test if this

speculation is correct. Despite these differences among the various

models, it is obvious that DS animal models have impaired

synaptic plasticity and, thus, an abnormal memory function. It

would be worthwhile to investigate further the mechanisms res-

ponsible for the changes in synaptic activity of BAC TG mice,

focusing especially on postsynaptic signal transduction.

Thus far, a mechanism-based drug to treat DS mental

retardation has not been developed. If the learning and memory

deficit phenotype resulting from DYRK1A over-production is a

reversible phenomenon, a small-molecule drug that inhibits the

kinase activity of DYRK1A might be effective in mitigating the

effects of excess DYRK1A. Because the learning and memory

ability of BAC TG mice is significantly impaired relative to control

mice, our new model system constitutes a powerful tool for testing

drug efficacy in vivo.

Acknowledgments

We thank J. H. Kang and D. E. Lee for technical support and

Dr. J. F. Cheng for help with the BAC clone search. Special thanks

go to Drs. W. Seol, S. H. Chung, and D. Kurnit for reviewing the

K.-J. Ahn et al. / Neurobiology of Disease 22 (2006) 463–472 471

manuscript. This work was supported by the IBST grant 2005

from Inje University and the Korea Research Foundation Grant

(R08-2004-000-10231).

References

Altafaj, X., Dierssen, M., Baamonde, C., Marti, E., Visa, J., Guimera, J.,

Oset, M., Gonzalez, J.R., Florez, J., Fillat, C., Estivill, X., 2001.

Neurodevelopmental delay, motor abnormalities and cognitive deficits

in transgenic mice overexpressing Dyrk1A (minibrain), a murine model

of Down’s syndrome. Hum. Mol. Genet. 10, 1915–1923.

Becker, W., Joost, H.G., 1999. Structural and functional characteristics of

Dyrk, a novel subfamily of protein kinases with dual specificity. Prog.

Nucleic Acid Res. Mol. Biol. 62, 1–17.

Branchi, I., Bichler, Z., Minghetti, L., Delabar, J.M., Malchiodi-Albedi, F.,

Gonzalez, M.C., Chettouh, Z., Nicolini, A., Chabert, C., Smith, D.J.,

Rubin, E.M., Migliore-Samour, D., Alleva, E., 2004. Transgenic

mouse in vivo library of human Down syndrome critical region 1:

association between DYRK1A overexpression, brain development

abnormalities, and cell cycle protein alteration. J. Neuropathol. Exp.

Neurol. 63, 429–440.

Costa, A.C., Grybko, M.J., 2005. Deficits in hippocampal CA1 LTP

induced by TBS but not HFS in the Ts65Dn mouse: a model of Down

syndrome. Neurosci. Lett. 382, 317–322.

Crabbe, J.C., Wahlsten, D., Dudek, B.C., 1999. Genetics of mouse

behavior: interactions with laboratory environment. Science 284,

1670–1672.

Demas, G.E., Nelson, R.J., Krueger, B.K., Yarowsky, P.J., 1998. Impaired

spatial working and reference memory in segmental trisomy (Ts65Dn)

mice. Behav. Brain Res. 90, 199–201.

Ema, M., Ikegami, S., Hosoya, T., Mimura, J., Ohtani, H., Nakao, K.,

Inokuchi, K., Katsuki, M., Fujii-Kuriyama, Y., 1999. Mild impairment

of learning and memory in mice overexpressing the mSim2 gene

located on chromosome 16: an animal model of Down’s syndrome.

Hum. Mol. Genet. 8, 1409–1415.

Escorihuela, R.M., Vallina, I.F., Martinez-Cue, C., Baamonde, C., Dierssen,

M., Tobena, A., Florez, J., Fernandez-Teruel, A., 1998. Impaired short-

and long-term memory in Ts65Dn mice, a model for Down syndrome.

Neurosci. Lett. 247, 171–174.

Fotaki, V., Dierssen, M., Alcantara, S., Martinez, S., Marti, E., Casas, C.,

Visa, J., Soriano, E., Estivill, X., Arbones, M.L., 2002. Dyrk1A hap-

loinsufficiency affects viability and causes developmental delay and

abnormal brain morphology in mice. Mol. Cell. Biol. 22, 6636–6647.

Gahtan, E., Auerbach, J.M., Groner, Y., Segal, M., 1998. Reversible

impairment of long-term potentiation in transgenic Cu/Zn-SOD mice.

Eur. J. Neurosci. 10, 538–544.

Galceran, J., de Graaf, K., Tejedor, F.J., Becker, W., 2003. The

MNB/DYRK1A protein kinase: genetic and biochemical properties.

J. Neural. Transm., Suppl., 139–148.

Guimera, J., Casas, C., Pucharcos, C., Solans, A., Domenech, A., Planas,

A.M., Ashley, J., Lovett, M., Estivill, X., Pritchard, M.A., 1996. A

human homologue of Drosophila minibrain (MNB) is expressed in the

neuronal regions affected in Down syndrome and maps to the critical

region. Hum. Mol. Genet. 5, 1305–1310.

Guimera, J., Casas, C., Estivill, X., Pritchard, M., 1999. Human minibrain

homologue (MNBH/DYRK1): characterization, alternative splicing,

differential tissue expression, and overexpression in Down syndrome.

Genomics 57, 407–418.

Hammerle, B., Carnicero, A., Elizalde, C., Ceron, J., Martinez, S., Tejedor,

F.J., 2003a. Expression patterns and subcellular localization of the

Down syndrome candidate protein MNB/DYRK1A suggest a role in

late neuronal differentiation. Eur. J. Neurosci. 17, 2277–2286.

Hammerle, B., Elizalde, C., Galceran, J., Becker, W., Tejedor, F.J., 2003b.

The MNB/DYRK1A protein kinase: neurobiological functions and

Down syndrome implications. J. Neural Transm., 129–137.

Harris-Cerruti, C., Kamsler, A., Kaplan, B., Lamb, B., Segal, M., Groner, Y.,

2004. Functional and morphological alterations in compound transgenic

mice overexpressing Cu/Zn superoxide dismutase and amyloid precursor

protein [correction]. Eur. J. Neurosci. 19, 1174–1190.

Hattori, M., Fujiyama, A., Taylor, T.D., Watanabe, H., Yada, T., Park, H.S.,

Toyoda, A., Ishii, K., Totoki, Y., Choi, D.K., Groner, Y., Soeda, E.,

Ohki, M., Takagi, T., Sakaki, Y., et al., 2000. The DNA sequence of

human chromosome 21. Nature 405, 311–319.

Hayashi, M.L., Choi, S.Y., Rao, B.S., Jung, H.Y., Lee, H.K., Zhang, D.,

Chattarji, S., Kirkwood, A., Tonegawa, S., 2004. Altered cortical

synaptic morphology and impaired memory consolidation in forebrain-

specific dominant-negative PAK transgenic mice. Neuron 42, 773–787.

Holtzman, D.M., Santucci, D., Kilbridge, J., Chua-Couzens, J., Fontana,

D.J., Daniels, S.E., Johnson, R.M., Chen, K., Sun, Y., Carlson, E.,

Alleva, E., Epstein, C.J., Mobley, W.C., 1996. Developmental abnor-

malities and age-related neurodegeneration in a mouse model of Down

syndrome. Proc. Natl. Acad. Sci. U. S. A. 93, 13333–13338.

Jacobs, P.A., Baikie, A.G., Court Brown, W.M., Strong, J.A., 1959. The

somatic chromosomes in mongolism. Lancet 1, 710.

Kentrup, H., Becker, W., Heukelbach, J., Wilmes, A., Schurmann, A.,

Huppertz, C., Kainulainen, H., Joost, H.G., 1996. Dyrk, a dual

specificity protein kinase with unique structural features whose activity

is dependent on tyrosine residues between subdomains VII and VIII. J.

Biol. Chem. 271, 3488–3495.

Kleschevnikov, A.M., Belichenko, P.V., Villar, A.J., Epstein, C.J., Malenka,

R.C., Mobley, W.C., 2004. Hippocampal long-term potentiation sup-

pressed by increased inhibition in the Ts65Dn mouse, a genetic model

of Down syndrome. J. Neurosci. 24, 8153–8160.

Korenberg, J.R., Kawashima, H., Pulst, S.M., Ikeuchi, T., Ogasawara, N.,

Yamamoto, K., Schonberg, S.A., West, R., Allen, L., Magenis, E., et al.,

1990. Molecular definition of a region of chromosome 21 that causes

features of the Down syndrome phenotype. Am. J. Hum. Genet. 47,

236–246.

Korenberg, J.R., Chen, X.N., Schipper, R., Sun, Z., Gonsky, R., Gerwehr,

S., Carpenter, N., Daumer, C., Dignan, P., Disteche, C., 1994. Down

syndrome phenotypes: the consequences of chromosomal imbalance.

Proc. Natl. Acad. Sci. U. S. A. 91, 4997–5001.

Lejeune, J., Gautier, M., Turpin, R., 1959. Study of somatic chromosomes

from 9 Mongoloid children. C. R. Hebd. Seances Acad. Sci. 248,

1721–1722.

Lisman, J.E., 2001. Three Ca2+ levels affect plasticity differently: the LTP

zone, the LTD zone and no man’s land. J. Physiol. 532, 285.

Lochhead, P.A., Sibbet, G., Morrice, N., Cleghon, V., 2005. Activation-loop

autophosphorylation is mediated by a novel transitional intermediate

form of DYRKs. Cell 121, 925–936.

Marti, E., Altafaj, X., Dierssen, M., de la Luna, S., Fotaki, V., Alvarez, M.,

Perez-Riba, M., Ferrer, I., Estivill, X., 2003. Dyrk1A expression pattern

supports specific roles of this kinase in the adult central nervous system.

Brain Res. 964, 250–263.

Martinez de Lagran, M., Altafaj, X., Gallego, X., Marti, E., Estivill, X.,

Sahun, I., Fillat, C., Dierssen, M., 2004. Motor phenotypic alterations in

TgDyrk1a transgenic mice implicate DYRK1A in Down syndrome

motor dysfunction. Neurobiol. Dis. 15, 132–142.

Migaud, M., Charlesworth, P., Dempster, M., Webster, L.C., Watabe, A.M.,

Makhinson, M., He, Y., Ramsay, M.F., Morris, R.G., Morrison, J.H.,

O’Dell, T.J., Grant, S.G., 1998. Enhanced long-term potentiation and

impaired learning in mice with mutant postsynaptic density-95 protein.

Nature 396, 433–439.

Morris, R.G., Willshaw, D.J., 1989. Memory. Must what goes up come

down? Nature 339, 175–176.

Morris, R.G., Garrud, P., Rawlins, J.N., O’Keefe, J., 1982. Place navigation

impaired in rats with hippocampal lesions. Nature 297, 681–683.

Nishiyama, H., Knopfel, T., Endo, S., Itohara, S., 2002. Glial protein S100B

modulates long-term neuronal synaptic plasticity. Proc. Natl. Acad. Sci.

U. S. A. 99, 4037–4042.

Okui, M., Ide, T., Morita, K., Funakoshi, E., Ito, F., Ogita, K., Yoneda, Y.,

Kudoh, J., Shimizu, N., 1999. High-level expression of the

K.-J. Ahn et al. / Neurobiology of Disease 22 (2006) 463–472472

Mnb/Dyrk1A gene in brain and heart during rat early development.

Genomics 62, 165–171.

Patterson, D., 1987. The causes of Down syndrome. Sci. Am. 257, 52–57

(60).

Paulsen, O., Sejnowski, T.J., 2000. Natural patterns of activity and

long-term synaptic plasticity. Curr. Opin. Neurobiol. 10, 172–179.

Pulsifer, M.B., 1996. The neuropsychology of mental retardation. J. Int.

Neuropsychol. Soc. 2, 159–176.

Rahmani, Z., Blouin, J.L., Creau-Goldberg, N., Watkins, P.C., Mattei, J.F.,

Poissonnier, M., Prieur, M., Chettouh, Z., Nicole, A., Aurias, A., et al.,

1989. Critical role of the D21S55 region on chromosome 21 in the

pathogenesis of Down syndrome. Proc. Natl. Acad. Sci. U. S. A. 86,

5958–5962.

Rahmani, Z., Lopes, C., Rachidi, M., Delabar, J.M., 1998. Expression of

the mnb (dyrk) protein in adult and embryonic mouse tissues. Biochem.

Biophys. Res. Commun. 253, 514–518.

Raich, W.B., Moorman, C., Lacefield, C.O., Lehrer, J., Bartsch, D.,

Plasterk, R.H., Kandel, E.R., Hobert, O., 2003. Characterization of

Caenorhabditis elegans homologs of the Down syndrome candidate

gene DYRK1A. Genetics 163, 571–580.

Reeves, R.H., Irving, N.G., Moran, T.H., Wohn, A., Kitt, C., Sisodia, S.S.,

Schmidt, C., Bronson, R.T., Davisson, M.T., 1995. A mouse model for

Down syndrome exhibits learning and behaviour deficits. Nat. Genet.

11, 177–184.

Sago, H., Carlson, E.J., Smith, D.J., Rubin, E.M., Crnic, L.S., Huang, T.T.,

Epstein, C.J., 2000. Genetic dissection of region associated with

behavioral abnormalities in mouse models for Down syndrome. Pediatr.

Res. 48, 606–613.

Saura, C.A., Choi, S.Y., Beglopoulos, V., Malkani, S., Zhang, D.,

Shankaranarayana Rao, B.S., Chattarji, S., Kelleher, R.J. III, Kandel,

E.R., Duff, K., Kirkwood, A., Shen, J., 2004. Loss of presenilin

function causes impairments of memory and synaptic plasticity

followed by age-dependent neurodegeneration. Neuron 42, 23–36.

Shindoh, N., Kudoh, J., Maeda, H., Yamaki, A., Minoshima, S.,

Shimizu, Y., Shimizu, N., 1996. Cloning of a human homolog of

the Drosophila minibrain/rat Dyrk gene from ‘‘the Down syndrome

critical region’’ of chromosome 21. Biochem. Biophys. Res.

Commun. 225, 92–99.

Siarey, R.J., Carlson, E.J., Epstein, C.J., Balbo, A., Rapoport, S.I.,

Galdzicki, Z., 1999. Increased synaptic depression in the Ts65Dn

mouse, a model for mental retardation in Down syndrome. Neurophar-

macology 38, 1917–1920.

Siarey, R.J., Villar, A.J., Epstein, C.J., Galdzicki, Z., 2005. Abnormal

synaptic plasticity in the Ts1Cje segmental trisomy 16 mouse model of

Down syndrome. Neuropharmacology 49, 122–128.

Smith, D.J., Stevens, M.E., Sudanagunta, S.P., Bronson, R.T., Makhinson,

M., Watabe, A.M., O’Dell, T.J., Fung, J., Weier, H.U., Cheng, J.F.,

Rubin, E.M., 1997. Functional screening of 2Mb of human chromosome

21q22.2 in transgenic mice implicates minibrain in learning defects

associated with Down syndrome. Nat. Genet. 16, 28–36.

Song, W.J., Sternberg, L.R., Kasten-Sportes, C., Keuren, M.L., Chung,

S.H., Slack, A.C., Miller, D.E., Glover, T.W., Chiang, P.W., Lou, L.,

Kurnit, D.M., 1996. Isolation of human and murine homologues of the

Drosophila minibrain gene: human homologue maps to 21q22.2 in the

Down syndrome ‘‘critical region’’. Genomics 38, 331–339.

Tejedor, F., Zhu, X.R., Kaltenbach, E., Ackermann, A., Baumann, A.,

Canal, I., Heisenberg, M., Fischbach, K.F., Pongs, O., 1995. minibrain:

a new protein kinase family involved in postembryonic neurogenesis in

Drosophila. Neuron 14, 287–301.

Winocur, G., Roder, J., Lobaugh, N., 2001. Learning and memory in

S100-beta transgenic mice: an analysis of impaired and preserved

function. Neurobiol. Learn. Mem. 75, 230–243.

Zeng, H., Chattarji, S., Barbarosie, M., Rondi-Reig, L., Philpot, B.D.,

Miyakawa, T., Bear, M.F., Tonegawa, S., 2001. Forebrain-specific

calcineurin knockout selectively impairs bidirectional synaptic plasticity

and working/episodic-like memory. Cell 107, 617–629.