Shinshu Med J, 69⑴：45～52, 2021

Inhibition of CASK Expression by Virus-mediatedRNA Interference in Medial Prefrontal Cortex Affects
Social Behavior in the Adult Mouse
Xueshan Cao1）†, Wen Qiu1）†, Bo Pang1）
Mengyun Zhou1）, Anuradha Mehta1）, Qi Guo1）
Yoshinori Shirai1）, Takuma Mori1）2） and Katsuhiko Tabuchi1）2）＊
1） Department of Molecular and Cellular Physiology, Shinshu University School of Medicine
2） Department of NeuroHealth Innovation, Institute for Biomedical Sciences,
Interdisciplinary Cluster for Cutting Edge Research, Shinshu University

Background : Calcium/calmodulin-dependent serine protein kinase (CASK) is a synaptic scaffolding protein andmutations in the CASK gene have been identified in various types of neurodevelopmental disorders. Deficit
in social behavior is a common symptom of many neurodevelopmental disorders and also accompanies CASK
related disorders. The deep layer of the medial prefrontal cortex (mPFC) has been suggested to be responsible
for social behavior.
Methods : To study the effect of CASK deficiency on social behavior, we generated mPFC specific CASK knockdown mice by introducing CASK short-hairpin RNA using adeno-associated viral (AAV) injection. We studied
the behaviors of CASK knockdown and enhanced green fluorescent protein (EGFP) expressing AAV injected
control mice using open field and three-chamber apparatus.
Results : CASK knockdown mice showed normal locomotor activity, anxiety level, and repetitive behavior in theobservation in the open field arena, but they showed deficit in reciprocal social interaction with a juvenile mouse.
In the three-chamber test, CASK knockdown mice showed more interaction with a novel juvenile mouse thanan empty cage at a similar level to control mice, suggesting that social recognition is intact. On the other hand,no significant difference was observed in the interaction between a novel target mouse and a pre-exposedmouse, suggesting that social memory ability was impaired in the CASK knockdown mice.
Conclusion : By using mPFC specific CASK knockdown mice, we found that CASK deficiency in this area
affects the social memory ability. Our results provide insights not only into CASK related disorders, but also awide range of neurodevelopmental disorders associated with social deficits. Shinshu Med J 69 : 45―52, 2021
(Received for publication September 4, 2020 ; accepted in revised form September 8, 2020)
Key words : CASK, neurodevelopmental disorder, medial prefrontal cortex, short-hairpin RNA,adeno-associated virus＊

Corresponding author : Katsuhiko Tabuchi
Department of Molecular and Cellular Physiology,
Shinshu University School of Medicine/Department ofIntroduction

Calcium/calmodulin-dependent serine protein ki-

NeuroHealth Innovation, Institute for Biomedical Sciences,

nase (CASK) is a family of membrane associated gua-

Interdisciplinary Cluster for Cutting Edge Research,nylate kinase protein (MAGUK) fundamentally local-

Shinshu University, 3-1-1 Asahi, Matsumoto, Nagano
390-8621, Japan
E-mail : ktabuchi@shinshu-u.ac.jp†

Ⅰ

Xueshan Cao and Wen Qiu contributed equally to this work

No. 1, 2021

ized at the pre- and postsynaptic terminals. CASK
protein consists of the CaMKII like domain, L27
domain, PDZ domain, SH3 domain, and guanylate45

Cao・Qiu・Pang et al.

kinase domain, each of which binds intracellular or

action, social recognition, and social learning andtransmembrane proteins that mediate signal trans-

found that the CASK knockdown mice exhibited

ductions required for the development and mainte-

deficits in social interaction and social learning. Our

1）nance of synapse . Mutations, deletions, and copy

results indicate that CASK deficiency in the mPFC isnumber variations of the CASK gene have been

responsible for the social deficits observed in theidentified in the patients with neurodevelopmental

patients with CASK mutation.

2）

disorders . The best known CASK-related disorder

Ⅱ Methods

is microcephaly with cerebellar and pontine hypoplasia (MICPCH) syndrome3）. MICPCH syndrome is anＡ Construction of adeno-associated viral (AAV)
vectors

X-linked neurodevelopmental disorder characterized
by a smaller brain, especially in the cerebellum, and
4）

pAAV-H1-shCASK/U6-hSyn-EGFP-WPRE :facial malformation . Social deficits, intellectual dis-

The shRNA target sequence for CASK, of which

ability, and infantile epilepsy accompany many cases

knockdown efficiency was verified previously6）7）, wasof the syndrome. Most of the patients with MICPCH

5’-ATCCATGAGCAGGGGCTGA-3’. shCASK oligos

syndrome are females, probably due to prenatal le-(KT14322 and KT14323) were annealed (95 ℃, 5min,thality in males. CASK knockout (KO) mice havethen slowly cooled down to room temperature) andbeen generated and studied. The male CASK KO

cloned into the XhoI/XbaI site of pAAV-H1/U6-

and homozygote female mice die shortly after birthhSyn-EGFP-WPRE.

5）

due to multiple organ failure . We previously studied

KT14322 : 5’-TCGACCCATCCATGAGCAGGGGCTG

heterozygote CASK female KO mice as models of

ATTCAAGAGATCAGCCCCTGCTCATGGATTTTMICPCH syndrome. We identified that the X chro-

TTTGGAAAT-3’

mosome inactivation is involved in the pathophysiol-

KT14323 : 5’-CTAGATTTCCAAAAAAATCCATG

ogy of MICPCH syndrome and the balance between

AGCAGGGGCTGATCTCTTGAATCAGCCCCTGCT

excitatory and inhibitory synapses projecting to the

CATGGATGGG-3’

CASK-deficient neurons was disrupted in the modelＢ

6）

AAV preparation
HEK293-AAV cells (10cm dish, 80 ％ confluent)

mice .

One major question for genetic neuropsychiatricwere co-transfected with pAAV plasmid (5ug),

disorder is the relevance between genes and the

pHelper plasmid (10ug) and pRC-DJ plasmid (5.5ug)

cause of behavioral symptoms. Social deficits are theusing polyethyleneimine (PEI). 3 days after transfec-

principal behavioral phenotype in most of the neuro-tion, cells were harvested, disrupted by repeateddevelopmental disorders. Protocols for evaluating

freeze-and-thaw (Liq. nitrogen/37 ℃ water bath, 4social behavior in mouse models have been well es-

times), centrifuged and virus-containing supernatanttablished. Open field and three-chamber tests are

was collected. The supernatant was treated with

commonly used for the purpose, but the major lim-

benzonase nuclease (0.125U/ul) at 37 ℃ for 30 min,itation is that females cannot be used for those kinds

then centrifuged and the supernatant was collectedof experiments. Pheromone exhaling from female

as the AAV solution. AAV solutions were titrated bymice affects the results of social behavioral studies.

q u a n t i t a t i v e P C R u s i n g p r i m e r s K T 18345 a n d

As mentioned, male CASK KO mice die shortly after

KT99100 for EGFP.

birth. To study the social behavior in CASK-deficient

KT18345 : 5’-AAGGGCGAGGAGCTGTTCAC-3’

mice, we established CASK knockdown male model

KT99100 : 5’-TGCAGATGAACTTCAGGGTC-3’

mice by introducing short-hairpin RNA for CASKＣ

AAV injection(shCASK) specifically in the medial prefrontal cortex

Wild-type C57B/6J males, deeply anesthetized(mPFC) where relevance to social behavior was sug-with a ketamine and xylazine cocktail (100mg/kg

gested. We studied the general activities, social inter-

B.W and 10mg/kg B.W., respectively, i.p.), were46

Shinshu Med J

ol. 69CASK knockdown affects social behavior

injected with AAV into the mPFC at postnatal 19-

10 min. The two side chambers contained an invert-or 20-days old on a stereotaxic device (Narshige).

ed empty small black wire cup. After the habitua-

The coordination of viral injections was 1.75 mm

tion, mice were placed back into the central chamber(anterior from bregma), 0.3 mm (lateral from theand the doors to the side chambers were closed. In

midline) and 1.75 mm (depth from the brain surface).

the sociability test, an unfamiliar male mouse (strang-

Approximately 0.5 μl of AAV was injected in theer 1, S1), that had no prior contact with the subject

mPFC of each hemisphere of the mouse. The AAV-

mouse, was placed in one of the two cups, and theninjected mice were examined in the following behav-

the doorways were unblocked. The location of S1 in

ioral tests at the age of 2-4 months old. Correct

the left or right side chambers was randomly alter-

injection was confirmed histologically by EGFP sig-

nated between trials. The test mouse’s behaviors

nals in the brain sections after the behavioral experi-

were recorded for 10 min using a CCD camera fromments.

the top. After the sociability test, the subject mouseＤ

Behavioral tests

was again confined in the central chamber. In the

１

Open field testsocial novelty preference test, a second unfamiliar

Each mouse was placed in the center of the cylin-

male mouse (stranger 2, S2) was enclosed in the cup,

drical open field apparatus (Φ 50 X 40 cm, OF-25M,

which was empty (E) in the sociability test, and the

Muromachi). Subject’s behaviors in the apparatus

doorways were again unblocked. In both tests, thewere recorded from above the apparatus using aamount of time that the subject head was in each

CCD camera (Canon, Japan). The video frame ratechamber was measured as “time in chamber” andwas 30 frames per second (fps). The test lasted 30

within a 2-cm distance of the wire cup was mea-

min. The travel distance, the time in the center, andsured as “contact time”. The video was analyzed on

the walking speed was analyzed with CompACT

idTracker and further analyzed on a custom pro-

VAS ver. 3, a video tracking system (Muromachi,gram written in R.

Japan). We manually measured the vertical activityＥ(rearing and leaning), and grooming using the video.
２

Juvenile interaction tests

Statistical analysis
All values are represented by the average of inde-

pendent experiments±SEM. Statistical significanceOne day before the test, subject and stimulus mice

was determined by Student’s t test. Statistical analy-

were individually placed into a test arena (25×25×

sis was performed in R. Statistical significance (＊p＜31 cm), and allowed to explore the arena for 10 min.

0.05, ＊＊p＜0.01, ＊＊＊p＜0.001) was indicated by aster-Juvenile objects were 3-4 week old male wild-typeisks.

C57BL/6J mice. On the test day, the test (AAV
injected) mouse and a juvenile object mouse wereⅢ

Results

placed together into the arena. Their behaviors were

To investigate the effect of CASK deficiency in therecorded for 10 min using a CCD camera. The timesocial behavior, we generated CASK knockdown

spent in social contact by the subjects were analyzed

mice specifically in the mPFC by bilateral injection

by a trained observer. Behaviors that were scored asof AAV expressing shRNA targeting the CASK genesocial contact included the followings : sniffing,in adult mice (Fig. 1). These mice were healthy with-grooming, contact with nose, and following.

out showing obvious physical abnormalities.

３

Sociability and social novelty preference tests

We first examined the general activities of these

The apparatus was a rectangular, three-chambered

mice in the open field arena. CASK knockdown micebox. The chamber was 20×40×25 cm and the divid-

exhibited reduction of travel distance compared toing walls were made from clear Plexiglas, with small

the EGFP expressing AAV injected control mice, but

openings (5×3 cm) allowing access into each cham-other parameters, such as walking speed, time spentber. The mouse was habituated in the entire box forin the center, vertical activity, and grooming timeNo. 1, 2021

47

Cao・Qiu・Pang et al.

ａｂFig. 1 CASK knockdown in the mPFC by AAV expressing shRNA(a) shRNA targeting the CASK gene was introduced into the AAV (pAAV-H1-U6-hSyn-EGFP-WPRE) undercontrol of the H1 promoter. (b) AAV was injected into mPFC of each hemisphere in postnatal 19- or 20-day
old mice, and AAV-injected mice were subjected to behavioral tests at the age of 2-4 months old.

were unchanged (Fig. 2), suggesting that the locomo-

down mice have the normal ability to recognize mice

tor activity, anxiety, repetitive behavior were normal

as a social target. In the second round experiment,

in CASK knockdown mice.

another novel juvenile mouse was confined in the

Next, we introduced juvenile target mice in theempty cage of the three-chamber (S2). In this condi-

open field arena and examined the reciprocal social

tion, previous S1 became a pre-exposed “familiar”

interaction during free moving conditions. Comparedmouse. Control mice showed more interest in theto the control mice, CASK knockdown mice showednovel S2 mouse than the pre-exposed S1 mouse andless interaction with juvenile target mice (Fig. 3),

spent more time around S2 than S1. In contrast,

suggesting that the sociability was decreased in

CASK knockdown mice spent similar time between

CASK knockdown mice.

S1 and S2, indicating the memory of the pre-To dissect the type of the social deficits, we employed three-chamber tests. In the first round test,
we examined the interaction of test mice (control or

exposed S1 mouse was impaired (Fig. 4 d-f).
ⅣDiscussion

CASK knockdown mice) with a novel juvenile mouse

In the present study, we generated mPFC-specific(S1) and empty cage (E1). Both control and CASKCASK knockdown mice to study the effect of CASK

knockdown mice interacted more with a novel juve-

deficiency on the social deficits observed in CASK-

nile mouse (S1) than the empty cage at a similar

related neurodevelopmental disorders. We found thatratio (Fig. 4 a-c), suggesting that the CASK knock-

the reciprocal interaction with juvenile mice and48

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ol. 69CASK knockdown affects social behavior

Fig. 2 Trajectories and graphs for open field tests(a) Representative images for moving trajectories of control (shCon) and CASK knockdown (shCASK) mice.
Summary graphs for the travel distance (b), time in the center (c), walking speed (d), vertical activity (e), andgrooming time (f). ＊P＜0.05, ns : not significant. Control : 10 animals, CASK knockdown ; 9 animals.

Fig. 3 Reciprocal interaction with juvenile mice(a) Top view of the interaction with juvenile mice in the open field arena. (b) Summary graph for interaction
time. ＊＊＊P＜0.001. Control : 8 animals, CASK knockdown : 7 animals.
No. 1, 2021

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Cao・Qiu・Pang et al.

Fig. 4 Heatmap images and graphs for three-chamber tests(a) Heatmap images (S1 vs E), contact time (b), and time in chamber (c) for the sociability test. (d)Heatmap images (S1 vs S2), contact time (e), and time in chamber (f) for social novelty test. ＊P＜
0.05, ＊＊P＜0.01, ＊＊＊P＜0.001, ns : not significant. Control : 13 animals, CASK knockdown : 9 animals.

50

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ol. 69CASK knockdown affects social behaviorsocial memory were significantly impaired in CASK

anxiety level. Reduction of GABAergic synaptic func-

knockdown mice. Travel distance was decreased,tion is suggested to be the major cause of anxiety.

whereas waking speed, vertical activity, time in the

GABAergic function may not be decreased by CASK

center, and grooming were unchanged in the CASK

knockdown, or other brain regions may be more im-knockdown mice.

portant for anxiety. Electrophysiological study is re-

While fully deleted CASK mutations are foundonly in females, hypomorphic mutations are still
8）

quired to verify these possibilities.
A wide range of social behavioral abnormalities

found in males . Pathogenicity of CASK mutation

are observed in the CASK knockdown model. Many

between male and female patients is likely to be dif-studies suggest that the deep layer of the mPFC isferent because of the X chromosome inactivation

the center of dictating this behavior11）. Although dis-effect. In female patients, CASK-intact and CASK-

tinct neuronal pathophysiology is undefined, altereddeficient neurons are distributed randomly in a mo-balance between excitatory and inhibitory synaptic

6）

saic pattern in the brain . In males, genomic condi-

function is reported in many animal models exhibit-

tions of the CASK gene should be consistent in all

ing social behavioral deficits12）. Further electrophysi-

neurons. Infection efficiency of AAV is known to be

ological study in this area is necessary to examine

enormous and empirically almost all neurons within

this possibility.

9）

the injection area are expected to be infected . Since

CASK has been shown to interact with various

CASK male KO is lethal, our study using an AAV-

proteins through its functional domains13）. The best

mediated CASK knockdown model is the firststudied interaction molecule is the presynaptic cell

observation of CASK effects on behavior.

adhesion protein neurexin1）. CASK binds neurexin

Vertical activity and grooming are known tothrough the PDZ domain and participates in the

represent repetitive behavior observed in autistic

trams-synaptic complex including neuroligins and10）cases . Neurocircuits in the deep layer of the mPFC

PSD-95. These molecules are shown to be strongly

have been shown to be one of the responsive areas

linked to autism14）. To address whether the neurex-

for the behavior11）. We failed to detect these changes

in/neuroligin pathway is involved in the pathophysi-

in our mice, suggesting that CASK is not involved inology of the social deficits in CASK knockdown micethe mPFC-dependent repetitive behavior. We also

will open the window to dissect molecular mecha-

did not detect changes in the time in the center innisms underlying the core symptoms of CASK-

the open field test. This parameter is used to assess

related neurodevelopmental disorders.

References
1） Hata Y, Butz S, Sudhof TC : CASK : a novel dlg/PSD95 homolog with an N-terminal calmodulin-dependent protein
kinase domain identified by interaction with neurexins. J Neurosci 8 : 2488-2494, 1996
2） Hayashi S, Uehara DT, Tanimoto K, et al : Comprehensive investigation of CASK mutations and other genetic
etiologies in 41 patients with intellectual disability and microcephaly with pontine and cerebellar hypoplasia(MICPCH). PLoS One 8 : e0181791, 20173） Najm J, Horn D, Wimplinger I, et al : Mutations of CASK cause an X-linked brain malformation phenotype withmicrocephaly and hypoplasia of the brainstem and cerebellum. Nat Genet 9 : 1065-1067, 2008
4） Moog U, Bierhals T, Brand K, et al : Phenotypic and molecular insights into CASK-related disorders in males.
Orphanet J Rare Dis 44, 20155） Atasoy D, Schoch S, Ho A, et al : Deletion of CASK in mice is lethal and impairs synaptic function. Proc Natl Acad
Sci U S A 7 : 2525-2530, 2007
6） Mori T, Kasem EA, Suzuki-Kouyama E, et al : Deficiency of calcium/calmodulin-dependent serine protein kinase
disrupts the excitatory-inhibitory balance of synapses by down-regulating GluN2B. Mol Psychiatry 7 : 1079-1092,
2019
No. 1, 2021

51

Cao・Qiu・Pang et al.
7） Jeyifous O, Waites CL, Specht CG, et al : SAP97 and CASK mediate sorting of NMDA receptors through a previously
unknown secretory pathway. Nat Neurosci 8 : 1011-1019, 20098） Seto T, Hamazaki T, Nishigaki S, et al : A novel CASK mutation identified in siblings exhibiting developmental
disorders with/without microcephaly. Intractable Rare Dis Res 3 : 177-182, 2017
9） Chen LY, Jiang M, Zhang B, Gokce O, Sudhof TC : Conditional Deletion of All Neurexins Defines Diversity of
Essential Synaptic Organizer Functions for Neurexins. Neuron 3 : 611-625 e4, 201710） Silverman JL, Yang M, Lord C, Crawley JN : Behavioural phenotyping assays for mouse models of autism. Nat Rev
Neurosci 7 : 490-502, 201011） Yizhar O, Fenno LE, Prigge M, et al : Neocortical excitation/inhibition balance in information processing and social
dysfunction. Nature 7363 : 171-178, 201112） Tabuchi K, Blundell J, Etherton MR, et al : A neuroligin-3 mutation implicated in autism increases inhibitory synaptictransmission in mice. Science 5847 : 71-76, 2007
13） Tabuchi K, Biederer T, Butz S, Sudhof TC : CASK participates in alternative tripartite complexes in which Mint 1
competes for binding with caskin 1, a novel CASK-binding protein. J Neurosci 11 : 4264-4273, 2002
14） Jamain S, Quach H, Betancur C, et al : Mutations of the X-linked genes encoding neuroligins NLGN3 and NLGN4 areassociated with autism. Nat Genet 1 : 27-29, 2003

（2020. 9. 4 received ; 2020. 9. 8 accepted）

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