瑞特氏症新穎模式小鼠之研究

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行政院國家科學委員會專題研究計畫成果報告

瑞特氏症新穎模式小鼠之研究

研究成果報告(精簡版)

計畫類別：個別型

計畫編號： NSC 100-2320-B-004-001-

執行期間： 100 年 08 月 01 日至 101 年 07 月 31 日

執行單位：國立政治大學神經科學研究所

計畫主持人：廖文霖

計畫參與人員：此計畫無其他參與人員

公開資訊：本計畫涉及專利或其他智慧財產權，2 年後可公開查詢

中華民國 101 年 12 月 18 日

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中文摘要：瑞特氏症 (Rett Syndrome, RTT) 是一種主要發生在女性幼

童的神經發育疾病。典型 RTT 患者在出生時多為正常，症狀

通常在出生後 6-18 個月逐漸顯現，主要包括運動技能退

化、語言能力缺失，接著出現手部絞動之刻板行為，行動困

難以及自閉特徵。然而，在 RTT 患者中有大約 25-32%在出生

後前 3 個月即出現癲癇發作，因此被歸類為「非典型

RTT」。研究發現，X 染色體上的甲基 CpG 結合蛋白 2

（MeCP2）基因突變發生在超過 95%的典型 RTT 病例中，卻只

發生在 20-40%的非典型 RTT 患者上，顯示非典型 RTT 可能

肇因於其他的遺傳因子。最近的研究發現，第五型細胞週期

類蛋白磷酸激酶(cyclin-dependent kinase-like 5,簡稱

CDKL5)基因的突變發生在許多非典型 RTT 患者上。CDKL5 基

因位於 X 染色體上，負責製造一種絲胺酸/蘇胺酸蛋白磷酸

激酶，於出生後早期大量表現於成熟神經細胞中，可負責調

控皮質神經元的樹突形成；但目前仍不知是否 CDKL5 的缺失

會導致非典型 RTT 的發生。本研究主要透過分析 Cdkl5 基因

剔除小鼠之行為表現，並配合偵測其腦中神經傳導素的含

量，建立一個非典型 RTT 之小鼠模式。目前發現 Cdkl5 基因

剔除小鼠表現出明顯的運動協調障礙，社交障礙，過動及刻

板行為增加，並伴隨有紋狀體中多巴胺分泌失調及基因表現

的異常。因此，在小鼠腦中 Cdkl5 基因的缺失的確會造成與

非典型 RTT 及自閉症相類似的症狀。此 Cdkl5 基因剔除小鼠

模式的建立，有潛力成為未來針對非典型 RTT 及自閉症相關

症狀篩選藥物之平台。

中文關鍵詞：非典型瑞特氏症；第五型細胞週期類蛋白磷酸激酶；自閉

症；過動；紋狀體

英文摘要： Cyclin-dependent kinase-like 5 (CDKL5) is a X-linked

gene encoding a putative serine-threonine kinase.

Mutations of CDKL5 have been implicated in many

neurodevelopmental disorders including atypical Rett

Syndrome (aRTT) and autism spectrum disorders (ASDs).

Here we present the autism-like behaviors and

aberrant dopamine distribution in the brains of mice

lacking CDKL5. The 4-5-week-old male Cdkl5-/y mice

showed hyperactivity measured by traveling longer

distance with faster speed than their wild-type

littermates in an open-field test. By testing with

elevated zero maze and accelerating rotarod, the

Cdkl5-/y mice showed reduced anxiety and impaired

motor coordination, respectively. When we monitored

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the home-cage behavior Cdkl5 mutants, they kept

digging the bedding material and showed enhanced

motor stereotypy. In a three-chamber social test, the

Cdkl5-/y mice demonstrated impaired social

interaction when they encountered the novel stranger.

To understand the neural basis of the behavioral

phenotypes, we investigated the dopamine level in the

striatum of Cdkl5-/y mice. Interesting, opposite

pattern of dopamine alteration along the

rostral-caudal axis of the striatum was found in the Cdkl5-/y

mice comparing with the wild-type littermate

controls. Together, our findings suggest that CDKL5

is required for dopamine-mediated motor control and

involved in pathogenesis of autistic and hyperactive

behaviors. (Supported by National Science Council,

Taiwan. NSC100-2320-B-004-001,

NSC101-2320-B-004-003-MY2)

英文關鍵詞： Atypical Rett Syndrome； CDKL5； Autism；

Hyperactivity； Striatum

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Studies of Rett Syndrome with a Novel Mouse Model:

Loss of CDKL5 disrupts dopamine distribution in the striatum and causes

autism-like behaviors and hyperactivity in mice

By Wenlin Liao

ABSTRACT

Cyclin-dependent kinase-like 5 (CDKL5) is a X-linked gene encoding a putative

serine-threonine kinase. Mutations of CDKL5 have been implicated in many neurodevelopmental

disorders including atypical Rett Syndrome (aRTT) and autism spectrum disorders (ASDs).

Here we present the autism-like behaviors and aberrant dopamine distribution in the brains of

mice lacking CDKL5. The 4-5-week-old male Cdkl5

-/y

mice showed hyperactivity measured

by traveling longer distance with faster speed than their wild-type littermates in an open-field

test. By testing with elevated zero maze and accelerating rotarod, the Cdkl5

-/y

mice showed

reduced anxiety and impaired motor coordination, respectively. When we monitored the

home-cage behavior Cdkl5 mutants, they kept digging the bedding material and showed

enhanced motor stereotypy. In a three-chamber social test, the Cdkl5

-/y

_{mice demonstrated}

impaired social interaction when they encountered the novel stranger. To understand the

neural basis of the behavioral phenotypes, we investigated the dopamine level in the striatum

of Cdkl5

-/y

_{mice. Interesting, opposite pattern of dopamine alteration along the rostral-caudal}

axis of the striatum was found in the Cdkl5

-/y

mice comparing with the wild-type littermate

controls. Together, our findings suggest that CDKL5 is required for dopamine-mediated

motor control and involved in pathogenesis of autistic and hyperactive behaviors. (Supported

by National Science Council, Taiwan. NSC100-2320-B-004-001,

NSC101-2320-B-004-003-MY2)

KEYWORDS: Atypical Rett Syndrome; CDKL5; Autism; Hyperactivity; Striatum

INTRODUCTION

Rett syndrome (RTT) is a progressive neurodevelopmental disorder that predominantly

affects females with the prevalence of about 1 in 10,000 births. Girls with classic RTT (cRTT)

develop normally in the first 6-18 months, followed by an onset of stereotypic hand

movements, seizures, growth arrest and subsequent regression in language and motor skills

(Hagberg et al., 1983;Chahrour and Zoghbi, 2007). Mutations in X-linked gene encoding

methyl CpG-binding protein 2 (MeCP2) have been identified in the majority of cRTT patients

(Amir et al., 1999). The MeCP2 protein selectively binds to methylated CpG dinucleotides in

mammalian genome and is believed to mediate transcriptional repression through interactions

with histone deacetylase and corepressor Sin3a (Jones et al., 1998;Nan and Bird, 2001) or

gene activation by interacting with cAMP-response element binding protein (CREB)

(Chahrour et al., 2008).

About 20~40% of the girls with RTT show atypical phenotypes (Hanefeld variant) which

are characterized by seizures in the first three months of life (infantile spasm), early-onset

encephalopathy, global developmental delay and severe mental retardation (Hagberg,

2002;Hagberg and Skjeldal, 1994). Mutations in the X-linked cyclin-dependent kinase-like 5

[CDKL5, OMIM #300203; also known as serine threonine kinase 9 (STK9)] gene have been

repeatedly identified in patients with the Hanefeld variant of RTT and other severe

neurodevelopmental disorders, including infantile spasms, West syndrome, early-onset

intractible epilepsy and autism (Scala et al., 2005;Tao et al., 2004;Kalscheuer et al.,

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2003;Weaving et al., 2004;Archer et al., 2006;Evans et al., 2005), raising a possibility that

mutations in CDKL5 may be responsible for pathogenesis of these developmental disorders.

The CDKL5 gene is located on chromosome Xp22, containing 21 exons and encoding a

putative serine-threonine kinase (Montini et al., 1998). It is highly expressed in the

mammalian brain, enriched in mature neurons, and strongly induced at early postnatal stages

with peak expression at postnatal day 14 (Rusconi et al., 2008;Chen et al., 2010). Within

neurons, CDKL5 is enriched in the nucleus, and the nuclear localization is determined by its

C-terminal domain (Mari et al., 2005;Bertani et al., 2006;Rosas-Vargas et al., 2008;Lin et al.,

2005). Etiologically, numerous CDKL5 mutations found in patients may result in the

impairment of CDKL5 nuclear localization and cause disease states of aRTT (Weaving et al.,

2005;Rosas-Vargas et al., 2008). It was noteworthy that CDKL5 may interact with MeCP2

and phosphorylate MeCP2, thus functioning in the same molecular pathway of MeCP2 (Mari

et al., 2005;Bertani et al., 2006). Another nuclear protein, DNA methytransferase 1 (Dnmt1),

is also found to interact with CDKL5, and the latter may modulate the function of Dnmt1

through phosphorylation of its N-terminal domain (Kameshita et al., 2008). Recently, it was

reported that the subcellular localization of CDKL5 varies in different brain areas and is

largely accumulated in the cytoplasmic fraction at early postnatal stages in mouse brain and

also in the neurites of cultured cortical neurons (Rusconi et al., 2008). Supporting of this,

CDKL5 has been implicated in the regulation of neuronal migration and dendritic arborization

both in cultured neurons and in the developing mouse cortex (Chen et al., 2010). Recently,

CDKL5 has also been implicated in stabilizing excitatory synapses in neurons from patients

(Ricciardi et al., 2012).

In sum, CDKL5 may be localized in both the nucleus and the cytoplasm and exert

distinct functions in different cellular compartments by interacting with different substrate

proteins. Mutations of CDKL5 may impair its subcellular localization and specific cellular

functions. To clarify how CDKL5 deficiency causes symptoms of aRTT and ASD, it is

imperative to develop and characterize an animal model of CDKL5 deficiency. In the present

study, we analyzed the phenotypes of a novel mouse model of Cdkl5 gene knockout with a

battery of behavioral assays and biochemical approaches.

RESULTS

Lack of CDKL5 protein in the brain of Cdkl5

-/y

mice

The strategy to develop Cdkl5 knockout mouse is illustrated in figure 1A. The exon 6 of

Cdkl5 gene was flanked by two loxP sites. Upon Cre-mediated recombination, a translational

frame shift occurs in exon 7, resulting in a premature termination codon (TAA) and early

truncation of CDKL5 protein with N-terminal 96 amino acids as the partial kinase domain

(K96X), thus is equivalent to a loss-of-function mutant (Wang et al., 2012). This gene

knockout strategy recapitulates a mutation identified in patients with aRTT symptoms (Archer

et al., 2006). Sequencing of Cdkl5 mRNA confirmed the lack of exon 7 in samples from

Cdkl5 knockout (Cdkl5

-/y

) brains (Wang et al., 2012). Western blot analysis of the brain

lysates from either wild-type (Cdkl5

+/y

, WT) or Cdkl5

-/y

mice with antibodies raised against

the C-terminal domain of CDKL5 confirmed the absence of full-length CDKL5 protein in the

mutants (Fig. 1B).

Impaired motor coordination in both neonates and young adults of Cdkl5

-/y

_mice

With these CDKL5 deficient mice in hands, we started to characterize their behavioral

phenotypes from early postnatal stages. We reasoned that, if seizures (infantile spasm)

happened in infancy of pups lacking CDKL5, their spontaneous motor activity would likely

be affected. Supporting this, severe hypotonia, motor dyspraxia and ataxia have been reported

in patients with CDKL5 mutations (Archer et al., 2006;Rosas-Vargas et al., 2008). We thus

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measured the turning latencies of surface righting (SR) and negative geotaxis (NG) behaviors

of Cdkl5 mutant and WT pups from postnatal day 4 (P4) to P10 (See Materials and Methods)

(Schneider and Przewlocki, 2005;Wagner et al., 2006). We found that the SR response

showed significant improvement over time both in WT and Cdkl5 mutants by P6. However,

the mutant mice tend to spend longer time to surface right from P4 to P6 (Fig 2A; p = 0.088

A

Figure 1. Loss of CDKL5 protein in

Cdkl5 knockout mice. (A) The strategy of

developing Cdkl5 knockout mouse. The lox sites flanking the exon 6 are indicated by the two yellow triangles. TAA, the premature termination codon. K96X, the mutation of lysine (K) to stop codon (X) at the N-terminal 96th amino acid of CDKL5 protein. (B) Western blot analysis of brain lysates from either wild-type (WT, +/y) or

Cdkl5-/y_{(KO) mice with antibodies raised}

against the C-terminal domain of CDKL5 confirms the absence of full-length CDKL5 protein in all tested brain regions from

Cdkl5-/y_{mice. NAc, nucleus accumbens;}

ST-m, middle striatum; CT-m, middle cortex; mPFC, medial prefrontal cortex; Hip, hippocampus; Cbll, cerebellum.

B

and 0.086 on P4 and P6, respectively, compared to WT), suggesting that the development of

motor control is delayed at this stage in Cdkl5 mutant mice. We also found similar motor

deficits in NG response at later stage (Fig. 2B). The WT pups exhibited evident improvement

in the speed of turning from P6 to P8 and reached the plateau state after P8. In contrast, the

Cdkl5 mutants showed no significant improvement in this period (Fig 2B; p = 0.013, 0.01 and

0.021 for P8, P9, P10, respectively, compared to WT).

A

B

C

Figure 2. Impaired motor coordination in both pups and young adults of Cdkl5-/y (KO) mice. The surface

righting reflex (A) and negative geotaxis (B) of Cdkl5-KO mice and their wild-type (WT) littermates were tested at the age of postnatal (P) 4 to 10 days. Delayed turning of the body to the direction against gravity was found in Cdkl5-KO pups during P8-P10. (C) The Cdkl5-KO mice show a constant impairment in motor coordination on an accelerating rotarod tested at 4-5 weeks old for five consecutive days. *, p < 0.05; **, p < 0.01; two-way ANOVA with Bonferroni post-hoc test. All data are presented as mean ± SEM.

The motor deficits were further manifested with rotarod assessment in mice lacking

CDKL5 at one month of age (Fig 2C). Both WT and mutant mice displayed gradual

improvements during the 5-day’s testing period, however, the mutants showed significantly

shorter latency to fall at every trial than WT control (p < 0.05 from day 1 to day 4, compared

to WT), suggesting that mice lacking CDKL5 are impaired in motor coordination. These

deficits in the development of limb and body movement observed in Cdkl5 mutant mice are

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consistent with the pre-weaning behaviors found in MeCP2 mouse models of RTT (Santos et

al., 2007;De Filippis et al., 2010); and our unpublished observation) and early symptoms seen

in infants with RTT (Burford, 2005;Einspieler et al., 2005).

Enhanced stereotypic rearing and digging behavior in Cdkl5

-/y

mice

We next analyzed another behavioral phenotype, stereotypy, which is commonly

observed in RTT patients (Temudo et al., 2007;Archer et al., 2006;Rosas-Vargas et al., 2008).

We measured the total duration of mice spent in rearing in an open field and found that Cdkl5

-/y

_{mice showed significantly increased rearing behavior (Fig 3A; 71.5 ± 7.9 in KO vs. 29.3 ±}

3.7 in WT, p = 0.0005). To further investigate the stereotypic behavior of Cdkl5

-/y

mice, we

single-housed the tested mice in new cages for 5-6 hours followed by videotaping for 3

minutes. The Cdkl5

-/y

mice showed pronounced digging behavior, characterized by

continuously mining the bedding materials with forehead and forelimbs and moving forward

at the same time. When we measured the percentage of time for digging within the last two

minutes of each video clip, we found it was much longer in Cdkl5

-/y

mice than that in WT

controls (Fig 3B; 45.15 ± 6.93% in KO vs. 1.25 ± 0.96% in WT, p = 0.00035), indicating that

loss of CDKL5 enhances stereotypic behaviors in mice.

A

B

_{Figure 3. Enhanced stereotypic}

behavior in Cdkl5

-/y

_mice.

Rearing (A) and digging (B)

behaviors were measured in

Cdkl5

-/y

_{(KO) mice and their WT}

littermate controls at the age of

*4-5 weeks old. ***, p < 0.001;*

student-t test. All data are

presented as mean ± SEM.

Impaired social preference and social recognition in Cdkl5

-/y

mice

The impaired motor coordination and enhanced stereotypy in mice lacking CDKL5

suggest other autistic features may be also prominent in these mice. We next examined the

sociability of these mice because social impairment is one of the core symptoms of autism

spectrum disorders. The male mice at the age of 4-5 weeks old were tested in a three-chamber

social test (Sankoorikal et al., 2006) for three sessions and their moving trajectories were

videotaped and analyzed (Fig. 4). After habituation in the chamber for 10 min (session I, Fig.

4 A and D), the wild-type mice spent longer time in the chamber containing a stranger mouse

(S1) during session II showing the social preference activity (Empty: 78.2 ± 8.0; S1: 175.2 ±

9.4; p < 0.001, n = 9; Fig. 4 B and E). When we add a novel stranger mouse (S2) in the other

chamber (session III), the tested WT mice exhibited their interest in S2 (i.e. social

recognition) and stayed longer time in the chamber containing S2 (S1: 96.2 ± 10.8; S2: 158.4

± 11.9; p < 0.01, n = 9; Fig. 4 C and F). By contrast, the Cdkl5

-/y

mice stayed similar time in

both the chambers during the session II (Empty: 96.7 ± 12.1; S1: 136.8 ± 14.9; p > 0.05, n =

7; Fig. 4 B’ and E) and session III (S1: 112.8 ± 22.8; S2: 137.8 ± 23.3; p > 0.05, n = 7; Fig. 4

C’ and F), indicating loss of CDKL5 causes impairment in both social preference and social

recognition. Since the Cdkl5

-/y

mice perform normal novel object recognition (see Material &

Methods, data not shown), the recognition impairment of Cdkl5

-/y

mice is most likely specific

to the stimuli with social implications.

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D

E

F

Figure 4. Impaired sociability in male Cdkl5-/y_{mice. The representative trajectories (A-C’) and time spent in}

different chambers (D-F) of the mice tested at the age of 4-5 weeks old in the habituation session (A, A’, D), social preference session (B, B’, E) and social recognition session (C, C’, F) of the three-chamber social task. The wild-type (WT) mice spent longer time staying in the chambers with a stranger mouse (S1) during the session II (B, E) and with the novel stranger (S2) during the session III (C, F). The Cdkl5-KO mice show impairment in both social preference (B’, E) and social recognition (C’, F) compared with their WT littermate mice. S1, stranger 1; S2, stranger 2. **, p < 0.01; ***, p < 0.001; student-t test. All data are presented as mean ± SEM.

Enhanced locomotor activity and reduced anxiety in Cdkl5

-/y

mice

In habituation session of the social test, we noticed that the Cdkl5

-/y

mice traveled

longer than wild-type mice. We therefore tested them in an open field to measure their

locomotor activity (Fig. 5A). The mice were tested for 16 minutes with videotaping by a

top-mounted camera in a dim-lit and soundproofed room. The movie clips of 3.5~15.5 minutes

were selected for analysis. We found that Cdkl5

-/y

mice traveled significantly longer distances

(164.1 ± 9.1% of WT, p < 0.001) with faster speeds (for average velocity: 132.4 ± 5.0% of

WT, p < 0.001; for maximal velocity: 128.2 ± 8.4% of WT, p < 0.05) than wild-type

littermate controls (Fig. 5A), while they display no thigmotaxis, a tendency to avoid the center

arena, indicating that mice lacking CDKL5 develop reduced anxiety (243.1 ± 56.1% of WT, p

< 0.05; Fig. 5A). To further confirm the state of anxiety, the mice were tested with the

elevated zero maze (Fig. 5B). The Cdkl5

-/y

mice stayed significantly longer time in the open

sectors compared to the wild-type mice (23.52 ± 2.12% in KO vs. 13.67 ± 1.17% in WT, p <

0.001), showing the reduced anxiety in Cdkl5

-/y

mice. Notably, the increased time spent in the

open sectors in Cdkl5

-/y

mice is very likely confounded by their stereotypic hyperactivity and

uncontrolled impulsive/explorative behavior for what the Cdkl5

-/y

mice may exhibit less

preference and spend less time in the closed sectors of the maze. We also tested the

depression level of the mice with forced swimming test. There was no significant difference

found between the Cdkl5

-/y

mice and their wild-type controls (70.58 ± 4.30% in KO vs. 78.33

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± 5.99% in WT, p > 0.05; Fig. 5C), suggesting no depression-like feature was detected in

Cdkl5

-/y

mice.

A

B

C

Figure 5. Enhanced locomotor activity and reduced anxiety in Cdkl5-/y mice. (A) The quantification of open

field activities indicate enhanced locomotor activity of Cdkl5-KO (Cdkl5-/y_{) mice. Total Dist., total traveled}

distance; Resting T%, percentage of time at resting; V-avg, average velocity of locomotion; V-max, maximal velocity of locomotion; Center T%, percentage of time in center arena. (B) The Cdkl5-KO mice spend more time in the open sectors showing reduced anxiety in the elevated zero maze. (C) There is no significant difference between the Cdkl5-KO mice and their WT controls in total struggling time of forced swimming test. *, p < 0.05; ***, p < 0.001; student-t test. All data are presented as mean ± SEM.

Altered dopamine content in the striatum of Cdkl5

-/y

mice

According to the motor deficits and hyperactivity found in Cdkl5-/y mice, we next

explore whether the dopamine content altered in their brains. The tissues of different brain

regions were harvested from the mice at the age of 4-5 weeks old, and dopamine content of

lysates was measured by high performance liquid chromatography (HPLC). Significant

increase of dopamine was found in the rostral striatum and nucleus accumbens, whereas no

change and even slight reduction of dopamine occur in the middle striatum and caudal

striatum, respectively (Fig. 6). In the ventral midbrain (VMB) where the majority of

dopaminergic neurons located, a trend of increased dopamine was found in the Cdkl5-/y mice

but the change yet reached to the significant level (Fig. 6). There was no difference found

between the Cdkl5-/y mice and their wild-type controls for the dopamine content in the cortex

on the top of middle striatum.

Ａ

Ｂ

Figure 6. Altered dopamine content in the striatum of Cdkl5-/y_{mice. (A) The representative traces of the}

dopamine levels measured in different brain regions of

Cdkl5-/y _{(red line) and wild-type (WT, blue line) mice}

by HPLC. (B) Increase of dopamine in the ST-r and NAc while decrease of dopamine in ST-c found in the brains of Cdkl5-/y_{mice. No significant change of}

dopamine level is found in the ST-m, VMB and CT-m. ST-r, rostral striatum; ST-m, middle striatum; ST-c, caudal striatum; NAc, nucleus accumbens; VMB, ventral midbrain; CT-m, middle cortex. ***, p < 0.001; student-t test. All data are presented as mean ± SEM.

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DISCUSSION

In the present study, we characterized Cdkl5 knockout mice with a battery of behavioral

assays and investigate the neurochemical phenotypes associated with Cdkl5 deficiency. We

found that mice lacking functional CDKL5 recapitulated key characteristic features of aRTT,

such as enhanced spontaneous stereotypy of forelimb and impaired motor coordination on

rotarod performance. The mutants showed the motor deficits in surface righting and negative

geotaxis from early postnatal age, and hyperactive locomotion in an open field at the age of

4-5 weeks old. The Cdkl4-5-KO mice performed dampened social preference and social

recognition in the three-chamber social test, while with normal performance in novel object

recognition. Along with hyperactivity found in these Cdkl5-KO mice, higher dopamine levels

in the rostral striatum were increased, which was right opposite of the results found in

hypoactive Mecp2-KO mice (Liao et al., paper under reviewing). Interestingly, a decreasing

gradient of the dopamine levels was found from the rostral to caudal striatum of Cdkl5-KO

mice, which is contrast to the increasing gradient of dopamine found in the striatum of

Mecp2-KO mice. In addition to changes of dopamine levels, the protein expression of the

striosomal marker, mu-opioid receptor 1 (MOR1), was decreased by 50% in the striatum of

Cdkl5-KO mice (data not shown). These findings suggested CDKL5 protein is required for

control of dopamine release and striosomal marker expression in the striatum, and contributes

to control of locomotion, stereotypy, motor coordination and social behavior. The Cdkl5-KO

mice provide a potential animal model to gain insights into the neuropathology of aRTT and

autism.

MATERIALS AND MATHODS

Animals: All mice were bred and housed in a room with constant temperature (at 22 ± 1°C) and humidity (65 ±

5 %) with a 12-hour light-dark cycle at National Cheng-Chi University. Food and water were freely available. The mutant mice (original 129SV x C57/B6 genetic background, provided by our collaborator) were maintained in C57BL/6 background by back-crossing with C57BL/6J male mice. Our Cdkl5 knockout mice have now backcrossed more than ten generations. All animal protocols were approved by the Animal Care and Use Committee at NCCU. Given that Cdkl5 is an X-linked gene, female heterozygotes will have a mosaic expression pattern of Cdkl5 due to random X-inactivation. For simplicity, we focused on studying hemizygous male mice in the present study. Female heterozygous mice were kept to maintain the mouse line and bred with wild-type C57/BL6 males to generate experimental cohorts [Cdkl5-/y_{(KO) and Cdkl5}+/y_(WT)].

Genotyping: The Cdkl5 knockout mice were established by deletion of genomic fragment containing exon 6

(Figure 1A; Wang et al., 2012). All the mice were genotyped by polymerase chain reaction (PCR) using REDExtract- N-AmpTM Tissue PCR Kit (Sigma). Briefly, the tail tissues of mice were collected at 3-4 weeks old and incubated in 100ul mixtures of extraction solution and tissue preparation solution (4:1) at 55 °C for 20 min, the lysates were denatured at 95 °C for 3 min, followed by 4 °C for 5 min, and then adding 80ul neutralization solution and mixing well. One microlitter of lysate was used for each PCR reaction. The primers for genotyping of Cdkl5 gene were FW CCACCCTCTCAGT AAGGCAG-3’), and RV (5’-GTCCTTTTGCCACTCAATTC-3’). The PCR amplification was first carried out at 94°C for 5 min followed by 35 cycles at 94°C for 30 sec, 64°C for 40 sec, and 72°C for 60 sec. An additional process at 72°C for 5 min was run at the end of the PCR reaction. The PCR products of 653 bp and 305 bp are corresponded to WT and mutant allele, respectively.

Preparation of the brain sample: To collect brain samples for analysis, the brains of Cdkl5 mutants and their

WT littermates were subject to be perfused by 4% paraformaldehyde (PFA) or harvested by region-specific tissue dissection following the behavioral testing. The fixed brains were analyzed by immunohistochemistry for protein localization, and the tissues were used for Western blotting for protein quantification or high-performance liquid chromatography (HPLC) for measurement of dopamine content.

Immunohistochemistry: Immunohistochemistry was performed as previously described (Liao et al., 2008) with

the following primary antibodies: MOR1 (1:1000, Millipore) and PVB (1:2000, Sigma). All the antibodies were rabbit polyclonal except the PVB (derived from mouse). Following incubation with primary antibody, sections were incubated with secondary antibody, the biotinylated goat-anti-rabbit/ mouse IgG or rabbit-anti- rat IgG (1:500), in 0.1 M PBS containing 1% normal goat serum at room temperature for 1.5-2 hours. The sections were then incubated for 1-1.5 hr with avidin-biotin- complex (1:400, Vector). The immunoreactivity was detected with the substrate of diaminobenzedine in the presence of 0.003N hydrogen peroxide. The immunofluorescence staining was performed in the similar way if needed.

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Immunoblotting

Brain tissues were harvested immediately from decapitated mice and homogenized by sonication within lysis buffer containing protease inhibitor (Amresco). Twenty-µg protein lysate was separated by polyacrylamide gel electrophoresis (10%, Bio-Rad) with 150 volts for 1.5 h and transferred onto a PVDF membrane (Millipore) by liquid electroblotting (Mini Trans-Blot Cell, Bio-Rad) with 350mA for 1 h. The membrane was incubated with the primary antibodies of rabbit anti-dopamine D2 receptor (Millipore, 1:1000) and mouse anti-beta-actin (Novus, 1:100,000) at 4 °C for 16 hours. Following incubation with peroxidase-conjugate goat-anti-rabbit or goat-anti-mouse secondary antibodies for 2 h at room temperature, the protein signals were detected by an enhanced chemo-luminescence reagent kit (ECL, Millipore) under a bio-image acquisition system (Xlite 200R, Avegene Life Science). The digitized images were quantified with Image J (NIH software).

Measurement of dopamine by HPLC

The brain tissue was extracted on ice with 110 µl perchloric acid containing 0.45mM sodium hydrosulfite. After homogenization by sonication, extracts were centrifuged at 14,000 rpm for 10 min at 4°C, and filtered with nylon syringe filters (0.22 µm, Millipore). The HPLC system was composed of a solvent delivery system with an autosampler and electrochemical flow cell (VT-03, Antec, Leyden, The Netherlands). Separations were performed using a C18 column (250mm*4.6mm, Grace Alltima) with mobile phase containing 100mM

NaH2PO4•H2O, 0.74mM Heptane-1-sulfonic acid sodium salt, 0.027mM EDTA, 2mM KCl and 10% methanol

(pH adjusted to 3 with phosphoric acid). The mobile phase was filtered with 0.22 µm membranes filter (Critical, Inc) and degas for 50min before use, and then pumped into the separation system at a microflow rate of 0.8 ml/min. For ST-R, ST-M, ST-C and NAc, sixty microliter of the lysate was placed in the vial for injection twice of 20µl by auto-sampler. The samples of CTX-M and VMB were injected once for 35µl. The retention times for DOPAC and dopamine were 10~11 min and 14~16 min, respectively, as calibrated by their pure compounds (Sigma, USA). The integrated areas of dopamine peaks were calculated automatically (Clarity, DataApex).

Behavioral testing:

- Surface Righting : Each mouse was placed on its back and gently held with all four limbs extended outward at

which time it was released. Time to right (turn) such that all four paws were touching the surface was recorded. A maximum score of 30 sec was recorded when the mouse failed to right in that period (Schneider and Przewlocki, 2005;Wagner et al., 2006). Mice were tested on P4–P8.

- Negative Geotaxis: Negative geotropism was tested on P4-P10 by placing the mouse on a mesh grid (Schneider

and Przewlocki, 2005;Wagner et al., 2006). Each mouse was placed facing downward along a 45° incline. Latency to turn 180° such that the head was facing upward along the incline was recorded with a maximum of 30 s for each trial. Negative geotaxis reflects motor development and activity.

- Accelerating rotarod task: The mutants and their WT littermate controls were used in this study. The mice

were briefly trained on the rod (PanLab) with constant speed of 4 rpm for 30 sec. Following 30min interval, the mice were tested with accelerating speed from 4 to 40 rpm within 5 min. Three testing trials were performed on each day with an inter-trial interval of 30 min for five consecutive days. The training session were performed only prior to the first testing trial of the first day. The times at which a mouse fell down from the rotating rod were recorded automatically by the stop-plate for each trial. The median values of falling latency for three trials of each day were used for statistic analysis (Liao et al., 2008).

- Three-chamber social test: This task was designed to monitor the sociability of mice and tested as described

(Ricceri et al., 2007;Moy et al., 2004). The social testing arena was a rectangular, three-chambered box. Each chamber was 20 ´40 ´ cm in size. Dividing walls were made from clear Plexiglas, with rectangular openings (35 cm x 35 mm) allowing access into each chamber. The test mouse was first placed in the middle chamber and allowed to explore for five minutes. The openings into the two side chambers were obstructed by plastic boxes during this habituation phase. After the habituation period, an unfamiliar C57BL/6J male mouse (stranger 1) that has no prior contact with the subject mouse was placed in one of the side chambers. The location of stranger 1 in the left vs. right side chamber was systematically alternated between trials. The stranger mouse was enclosed in a small (60 mm x 60 mm x 100 mm), round plastic cylinder, which allows nose contact through the bars but prevented fighting. The animals serving as strangers had previously been habituated to placement in the small cylinder. An identical empty plastic cylinder was placed in the opposite chamber. Both openings to the side chambers were then unblocked and the subject mouse was allowed to explore the entire social test arena for a 10 min session. The amount of time spent in each chamber and the number of entries into each chamber were recorded by the Smart video-tracking system (PanLab, Spain).

- Novel object/place recognition: Novel object recognition and novel place recognition were tested according to

the modified protocol (Southwell et al., 2009). Briefly, mice were placed in the lower left corner of a 40 x 40 cm

open transparent Plexiglas box with 25 cm walls in a room brightly lit by fluorescent ceiling lights. Open field activity was recorded for 10 min by a ceiling-mounted video camera. The mice with skewed locomotion were excluded in the following test. During the 5 min inter-trial-interval (ITI), two different novel objects were placed in the upper two corners of the box, far enough (~7 cm) from the walls so as to not impede movement of testing animals. Mice were reintroduced to the box in the lower left corner and recorded for 10 min, during which the number of investigations of the two objects was scored. Episodes involving the mouse in close proximity to the objects but not facing or sniffing them were not considered investigations. Circling or rearing on the objects with continued sniffing was considered as a single investigation, while episodes in which the mouse sniffs the object, turns away, or rears against the wall and subsequently turns back to sniff the object again were considered separate investigations. Mice were then removed from the box for a 5 min ITI, and the object at the top right corner (the target object) of the box was replaced with a different, unfamiliar object in the

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same location. Mice were reintroduced to the box and recorded for 10 min, and the number of investigations of the objects was scored. For “novel object recognition test”, the percentage of the investigations of the target object (the unfamiliar one) was measured. For “novel place recognition test”, thetarget object was moved to the lower right corner of the box, and the mice were reintroduced into the box following an ITI of 5min. The percentage of the investigations or nose pokes to the target object (the one in the new location) was counted.

The recognition index was calculated as RI (%) = Rnew * 100% / (Rnew + Rold), where Rnew is the

number of investigation for the new or moved object, and Rold is the number of investigation for the familiar or unmoved object.

- Open-field activity: The mice (4-5 weeks old) were tested and videotaped within a clear Plexiglas

(40×40×25cm) open-field arena for 16 minutes with dim light in a sound-reduced testing room. The mouse activities (total traveled distance, percentage of resting time, average moving speed, maximal moving speed and percentage of time spend in the center) were analyzed for 12 mins (from 3.5’ ~ 15.5’) with the Smart® tracking System (Harvard, USA) excluding the first 3.5 mins of habituation time. The performance of mice of all genotypes was normalized with the average performance of wild-type mice. The mean and SEM were calculated and the differences between genotypes were compared by student-t test.

- Elevated zero maze: This task was designed to monitor the levels of anxiety of mice (Shepherd et al., 1994).

The apparatus consists of a circular platform (6cm width with a 45cm inner diameter) that is equally divided into four quadrants. Two opposite quadrants are enclosed by walls (15cm high), the other two quadrants are opened and bordered by a lip (0.6 cm high). The maze was elevated 40 cm above the tabletop and an overhead camera was activated to videotape the activity of the mouse on the maze. Each mouse was tested on the maze for 5 minutes and the time it stayed in the opened and closed arm was measured.

- Forced swimming test: The test was conducted as described (Porsolt et al., 2001). Briefly, mice were

individually forced to swim in an open cylindrical container (diameter 15 cm, height 22 cm), containing 18 cm of water at 25 ± 1 °C. The total duration of immobility measured during the last 4 min of the 6-min videotaping period. Each mouse was judged to be immobile when it ceased struggling and remained floating motionless in the water, making only those movements necessary to keep its head above water for at least 2 sec. They were removed and dried with a towel after the procedure. The water was changed after each test.

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國科會補助計畫衍生研發成果推廣資料表

日期:2011/11/24

國科會補助計畫

計畫名稱: 瑞特氏症新穎模式小鼠之研究計畫主持人: 廖文霖計畫編號: 100-2320-B-004-001- 學門領域: 醫學之生化及分子生物

無研發成果推廣資料

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100 年度專題研究計畫研究成果彙整表

計畫主持人：

廖文霖

計畫編號：

100-2320-B-004-001-計畫名稱：