(2023) 17:4
Kashii et al. Human Genomics
https://doi.org/10.1186/s40246-023-00450-2

Human Genomics

Open Access

RESEARCH

Tsc2 mutation rather than Tsc1 mutation
dominantly causes a social deficit in a mousemodel of tuberous sclerosis complex
Hirofumi Kashii1,2†, Shinya Kasai1†, Atsushi Sato1,3, Yoko Hagino1, Yasumasa Nishito4, Toshiyuki Kobayashi5,
Okio Hino5, Masashi Mizuguchi6 and Kazutaka Ikeda1*

Abstract
Background Tuberous sclerosis complex (TSC) is an autosomal dominant disorder that is associated with neurological symptoms, including autism spectrum disorder. Tuberous sclerosis complex is caused by pathogenic germline
mutations of either the TSC1 or TSC2 gene, but somatic mutations were identified in both genes, and the combined
effects of TSC1 and TSC2 mutations have been unknown.
Methods The present study investigated social behaviors by the social interaction test and three-chambered sociability tests, effects of rapamycin treatment, and gene expression profiles with a gene expression microarray in Tsc1 andTsc2 double heterozygous mutant (TscD+/−) mice.
Results TscD+/− mice exhibited impairments in social behaviors, and the severity of impairments was similar toTsc2+/− mice rather than Tsc1+/− mice. Impairments in social behaviors were rescued by rapamycin treatment in all
mutant mice. Gene expression profiles in the brain were greatly altered in TscD+/− mice more than in Tsc1+/− andTsc2+/− mice. The gene expression changes compared with wild type (WT) mice were similar between TscD+/− andTsc2+/− mice, and the overlapping genes whose expression was altered in mutant mice compared with WT mice
were enriched in the neoplasm- and inflammation-related canonical pathways. The “signal transducer and activator oftranscription 3, interferon regulatory factor 1, interferon regulatory factor 4, interleukin-2R α chain, and interferon-γ”
signaling pathway, which is initiated from signal transducer and activator of transcription 4 and PDZ and LIM domain
protein 2, was associated with impairments in social behaviors in all mutant mice.
Limitations It is unclear whether the signaling pathway also plays a critical role in autism spectrum disorders notcaused by Tsc1 and Tsc2 mutations.
Conclusions These findings suggest that TSC1 and TSC2 double mutations cause autistic behaviors similarly to TSC2
mutations, although significant changes in gene expression were attributable to the double mutations. These findings
contribute to the knowledge of genotype–phenotype correlations in TSC and suggest that mutations in both the
TSC1 and TSC2 genes act in concert to cause neurological symptoms, including autism spectrum disorder.
Keywords Double mutations, Pdlim2, Rapamycin, Tsc1, Tsc2, Tuberous sclerosis complex†

Hirofumi Kashii and Shinya Kasai contributed equally to this study.

*Correspondence:
Kazutaka Ikeda
ikeda-kz@igakuken.or.jp
Full list of author information is available at the end of the article© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
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Kashii et al. Human Genomics(2023) 17:4

Background
Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder that is characterized by twocore behavioral symptoms: deficits in social communication and social interaction, and restricted, repetitive
patterns of behaviors, interests, or activities [1]. Despite
its high clinical and etiological heterogeneity, recent
genetic studies have revealed that ASD appears to shareneurobiological mechanisms and be attributable to thedysregulation of some convergent pathways [2–5]. One
such pathway is the mechanistic target of rapamycincomplex 1 (mTORC1) signaling cascade [6–9].
Tuberous sclerosis complex (TSC), a prototypical mTORopathy, is an autosomal dominant disorder.
Up to 50% of patients with TSC have comorbid ASD.
The prevalence of TSC in overall ASD was estimatedto be 0.01 [10–12]. Thus, TSC is one of the most common diseases that is responsible for syndromic ASD.
Tuberous sclerosis complex is considered to be causedby pathogenic mutations in the TSC complex subunit
1 (TSC1) or TSC2 gene and is unique in the similarityof clinical phenotypes between pathogenic mutationsin the two genes [13–15]. TSC1 and TSC2 proteins
form a complex with complemental TBC domain family
member 7, which suppresses a small guanosine triphosphate-binding protein, Rheb, and negatively regulates
the mTORC1 signaling cascade [16]. The excessive activation of mTORC1 signaling that is caused by pathogenic mutations in the two genes is assumed to be theprimary pathophysiological mechanism of TSC [17].
According to Knudson’s two-hit theory, TSC-associatedtumors develop because of biallelic inactivation of the
TSC1 or TSC2 gene [18]. Indeed, the loss of heterozygosity (LOH) of either gene is often detected in most
TSC-associated tumors, and more than half of brain
cortical tubers lack LOH [19–21], suggesting that theheterozygosity of TSC1 or TSC2 is sufficient to cause
neuronal abnormalities [22].
Genotype–phenotype correlation studies suggest that
TSC2 pathogenic mutations rather than TSC1 pathogenic mutations are associated with severe phenotypic
manifestations, including early-onset seizures, a highrate of complications that are associated with ASD, lowcognitive function, and a high number of renal angiomyolipomas [15, 23–26]. Somatic TSC1 mutations wereidentified in lesions from patients with a pathogenic germline TSC2 mutation, suggesting that TSC1/TSC2 double heterozygous mutations may promote tumorigenesis[21, 27, 28]. Therefore, evaluations of manifestations ofdouble heterozygous pathogenic mutations comparedwith mutations of either gene alone will provide insights
into the pathophysiology of TSC. However, previous
studies exclusively focused on pathogenic mutations

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of either gene alone, and few studies have described
TSC1/TSC2 double mutations [29–31].
The homozygous loss of either Tsc1 or Tsc2 in micecauses embryonic lethality [32, 33]. Conventional heterozygous mice recapitulate some neuropsychiatricphenotypes of TSC in humans, including learning andmemory deficits and social impairments without apparent brain lesions [34–36]. Additionally, transient treatment with an mTORC1 inhibitor successfully rescued
these deficits.
The present study focused on the impact of Tsc1 andTsc2 double heterozygous mutations in mouse models. We analyzed social behaviors and gene expression
profiles in Tsc1 and Tsc2 double heterozygous mice andassessed the therapeutic effects of the mTOR inhibitor
rapamycin on autistic-like behaviors.

Materials and methods
Mice

Tsc1 heterozygous knockout (Tsc1+/−) and Tsc2 heterozygous knockout (Tsc2+/−) mice were generated
and maintained on a C57BL6/J background as previously described [32, 33]. A Tsc1+/− mouse and a Tsc2+/−
mouse were crossed to produce Tsc1+/+/Tsc2+/+ (WT)
mice, Tsc1+/−/Tsc2+/+ (Tsc1+/−) mice, Tsc1+/+/Tsc2+/−(Tsc2+/−) mice, and Tsc1+/−/Tsc2+/− (TscD+/−) mice.
C57BL/6J mice were purchased from CLEA Japan(Tokyo, Japan). After weaning at 3 weeks of age, all mice
were maintained separately by sex (two to five mice percage) in ventilated racks at 22 °C ± 2 °C under a 12 h/12 hlight/dark cycle (lights on 8:00 A.M. to 8:00 P.M.) withaccess to a standard laboratory diet and water ad libitum.
Drug

Rapamycin (LC Laboratories, Woburn, MA, USA) was
dissolved to 0.5 mg ­ml−1 with 10% dimethylsulfoxide(vehicle) and administered intraperitoneally in a volumeof 10 ml ­kg−1 once daily for 2 days. The behavioral tests
and brain collection for gene expression analysis began
24 h after the second rapamycin administration.
Behavioral tests

The hole board test, self-grooming test, social interaction test, three-chambered sociability test, elevated plus
maze test, and social transmission of food preference test
were performed with male and female mice at 14.4–21.6,
44.4–46.9, 17.1–43.4, 15.6–43.4, 15.6–26.1, and 16.0–
21.7 weeks of age between 10:00 A.M. and 4:00 P.M.,
respectively. The number of WT, Tsc1+/−, Tsc2+/−, andTscD+/− mice that were used in the behavioral tests were
13–19, 14–20, 15–20, and 14–22, respectively.
Hole board test Each mouse was placed in a hole board
apparatus (500 mm × 500 mm × 400 mm; Muromachi

Kashii et al. Human Genomics(2023) 17:4

Kikai, Tokyo, Japan) with four holes (38 mm diameter)
and allowed to freely explore it for 30 min. Behavior was
automatically analyzed for counts of head dipping using avideo tracking system.
Self-grooming test Each mouse was individuallyplaced in a new cage without bedding material. Afteran initial 10-min habituation period, the animal wasvideo-recorded for 10 min. The number of episodes ofself-grooming and rearing behavior was measured.
Social interaction test Each mouse was left alone inits home cage for 15 min. A novel mouse (C57BL/6J) of
the same sex and size was then introduced to the cage.
The behavior of the test mouse was video recorded for
10 min. The total duration of active interaction (anogenital sniffing, allogrooming, close following, and mounting)
was measured.
Three-chambered sociability tests A three-chamberarena (500 mm × 500 mm × 400 mm) was used to evaluate social approach, preference for social novelty, andsocial preference. After a 10 min habituation period, an
unfamiliar C57BL/6J mouse of the same sex (stranger1) was introduced to the columnar wire cage (100 mm
diameter, 100 mm height) in a side chamber, and a metal
block (object) was placed in another cage on the otherside chamber. The test mouse was allowed to explore
the entire arena for 10 min (Session 1: social approach).
The block was then replaced with another unfamiliar
mouse (stranger 2), and the test mouse was examined
for an additional 10 min (Session 2: social novelty). The
test mouse was then presented with another unfamiliar mouse (stranger 3) and a cagemate of the test mousefor 10 min (Session 3: social preference). The amount oftime spent around each cage was automatically measured
using a video tracking system. The approach-avoidancescore was calculated as the following: (time exploring
stranger 3) − (time exploring cagemate).
Elevated plus maze test Each mouse was individuallyplaced in the center of the elevated plus maze apparatus (Muromachi Kikai) and allowed to freely explore theapparatus for 10 min. Total distance traveled and timespent on the open arms were automatically measured
using a DV-Track Video Tracking System (Muromachi
Kikai).
Social transmission of food preference test Twentyfour hours before testing, the test mouse was placedin a novel cage with a specially designed food jar thatwas filled with powdered chow. Eighteen hours before
testing, the feeding jars were removed, and the mice
were deprived of food until testing. To begin testing,a “demonstrator” mouse was given a food jar that wasfilled with powdered chow that was flavored by mixing it with either 1% ground cinnamon or 2% powdered cocoa. After 1 h, the food jar was removed andPage 3 of 13

weighed to ensure that at least 0.2 g of food had been
consumed. The demonstrator mouse was then immediately placed in the cage of the “observer” test mouse,
and interactions between the demonstrator mouse andobserver mouse were allowed for 30 min. The demonstrator mouse was then removed, and two jars of
powdered food (one of each flavor) were placed in thecage with the observer mouse for 1 h. At the end of
the 1 h choice session, the food jars were removed andweighed. The demonstrator mice were C57BL/6J micethat were matched to the test observer mouse according to weight and sex.
Whole transcriptome analysis

Total RNA was isolated from the right brain hemisphereof 12-week-old male mice using Sepasol-RNA I Super G(nacalai tesque, Kyoto, Japan) and purified with RNeasy
Mini (Qiagen, Tokyo, Japan). Only RNA with an RNA
Integrity Number above 8.0, determined by an Agilent
Bioanalyzer (Agilent Technologies, Tokyo, Japan), wasused for gene expression analysis. Cy3-labeled cRNA wasprepared using the Low Input Quick Amp Labeling Kit(Agilent Technologies) according to the manufacturer’s
protocol, hybridized to the SurePrint G3 Mouse Gene
Expression v2 8 × 60 K Microarray (Agilent Technologies), and scanned using a SureScan Microarray Scanner(Agilent Technologies).
Microarray data were measured using Feature Extraction software (Agilent Technologies) and analyzed using
GeneSpring GX software (Agilent Technologies). Differentially expressed transcripts (DETs) were identified
by two-way analysis of variance (ANOVA) with p < 0.05.
Common transcripts between more than two DETs wereselected instead of multiple testing correction to minimize false positives. Clustering analysis was performed
using hierarchical clustering analysis with the default
Euclidean distance metric and default Ward’s linkage
rule.
Gene ontology and pathway enrichment analyses

Gene ontology (GO) and pathway enrichment analyses
were performed using MetaCore version 6.31 (https://​portal.​genego.​com/​cgi/​data_​manag​er.​cgi; accessed July22, 2022). Map Folders and GO Process Networks aremainly based on biochemical and signaling cascades andcellular processes, respectively. The statistical significance value and false discovery rate (FDR) were used torank GO. Canonical pathway modeling was used to identify pathways with statistical significance, zScore, andgScore by considering the number of objects of datasetsand canonical pathways in the networks.

Kashii et al. Human Genomics(2023) 17:4

Statistical analysis

The statistical analyses of behavioral data were performed using SPSS Statistics 24 software (IBM Japan
Ltd., Tokyo, Japan). Motor, sensory, and social behaviors
between mutant and WT mice were statistically evaluated using one-way ANOVA followed by the Tukey Honestly Significant Difference (HSD) test. Unpaired t-tests
were used to evaluate differences between vehicle andrapamycin treatment in the social interaction test. Paired
t-tests were used to evaluate differences between familiar and stranger mice in the three-chambered sociability
tests and between cued food and non-cued food in thesocial transmission of food preference test. Values ofp < 0.05 were considered statistically significant.

Results

Autistic‑like behaviors in ­Tsc1+/−, ­Tsc2+/−, and ­TscD+/−mice

The homozygous knockout of either the Tsc1 or Tsc2 genecauses embryonic lethality in mice, whereas both Tsc1+/−and Tsc2+/− mice are apparently healthy, do not die prematurely, and lack apparent cerebral lesions and spontaneous
seizures [32, 33]. TscD+/− mice were born at the expected

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Mendelian frequency and exhibited no apparent spontaneous seizures, no early death, and no delay of physicaldevelopment (data not shown). Adult Tsc1+/− and Tsc2+/−
mice have been reported to exhibit normal motor and sensory behaviors [36]. Similar to Tsc1+/− and Tsc2+/− mice,
TscD+/− mice did not display any significant differences inmotor or sensory behaviors in the hole board test (Fig. 1A),
elevated plus maze test, or self-grooming test comparedwith WT mice (Additional file 1: Fig. S1A–C). TscD+/−
mice normally discriminated the scent of a food that waspresented by stranger mice in the social transmission offood preference test, similar to Tsc1+/− and Tsc2+/− mice,indicating that these mutant mice can differentiate novel
nonsocial stimuli that are transmitted through social interaction (Additional file 1: Fig. S1D). TscD+/− mice exhibited
more frequent self-grooming behavior than WT mice,similar to Tsc1+/− and Tsc2+/− mice (p = 0.024, 0.031, and0.042 for Tsc1+/−, Tsc2+/−, and TscD+/− mice, respectively,
Tukey HSD test; Fig. 1B). TscD+/− mice also exhibited a
reduction in interaction time compared with WT mice,similar to Tsc1+/− and Tsc2+/− mice, in the social interaction test (p = 0.001, 0.004, and 0.004 for Tsc1+/−, Tsc2+/−,and TscD+/− mice, respectively, Tukey HSD test; Fig. 1C).

Fig. 1 Social behaviors in Tsc1+/−, Tsc2+/−, and TscD+/− mice. A–C Frequencies of head-dipping (A) and self-grooming (B) and time spent in active
interaction behaviors (C) in Tsc1+/−, Tsc2+/−, and TscD+/− mice in the hole board test, self-grooming test, and social interaction test, respectively.
D Time spent exploring (social approach) a stranger mouse (S1) and non-social object (O) in the three-chamber test. E Time spent exploring afamiliar mouse (S1) and stranger mouse (S2) in the social-novelty preference test. F Time spent exploring a cagemate (C) and stranger mouse (S3)in the social preference test. G Approach avoidance score calculated as the following: (time exploring S3) – (time exploring C). *p < 0.05; **p < 0.01;***p < 0.001, one-way ANOVA followed by Tukey HSD test (A–C, G) and paired t-test (D–F). Each bar indicates the mean ± SEM

Kashii et al. Human Genomics(2023) 17:4

In the social approach session of the three-chamberedsociability test, TscD+/− mice exhibited normal social preference, with a significant difference between the novel
mouse and non-social object, similar to WT, Tsc1+/−, andTsc2+/− mice (Fig. 1D). In the social novelty session, WT
mice spent significantly more time exploring the novel
mouse than the familiar mouse, whereas TscD+/− mice
exhibited no significant preference for social novelty, similar to Tsc1+/− and Tsc2+/− mice (p = 0.005, 0.581, 0.099,
and 0.845 for WT, Tsc1+/−, Tsc2+/−, and TscD+/− mice,
respectively, paired t-test; Fig. 1E). In the social preference session, TscD+/− mice spent a similar amount of time
exploring both the novel mouse and the cagemate, similar
to Tsc2+/− mice, whereas WT and Tsc1+/− mice preferred
the novel mouse significantly more than the cagemate(p < 0.001, p < 0.001, p = 0.725, and p = 0.422 for WT,
Tsc1+/−, Tsc2+/−, and TscD+/− mice, respectively, paired
t-test; Fig. 1F). The calculated approach-avoidance scorerevealed a severe reduction of social preference for a novel
mouse in Tsc2+/− and TscD+/− mice rather than Tsc1+/−
mice (p = 0.045, p < 0.001, p < 0.001 for Tsc1+/−, Tsc2+/−,and TscD+/− mice, respectively, compared with WT mice;p = 0.015, Tsc1+/− mice compared with Tsc2+/− mice,
Tukey HSD test; Fig. 1G).
Rapamycin ameliorated impairments in social behaviors
in ­TscD+/− mice similarly to ­Tsc1+/− and ­Tsc2+/− mice

We previously reported that 5–10 mg ­kg−1 rapamycin
ameliorated impairments in social interaction in Tsc1+/−and Tsc2+/− mice in the social interaction test [36]. The
time spent engaged in active interaction was increased by
5 mg ­kg−1 rapamycin in TscD+/− mice in the social interaction test similarly to Tsc1+/− and Tsc2+/− mice, with noeffect in WT mice (p = 0.559, 0.012, 0.016, and 0.001 for
WT, Tsc1+/−, Tsc2+/−, and TscD+/− mice, respectively,
unpaired t-test; Fig. 2A). Rapamycin also increased social
novelty preference in Tsc1+/− mice (p = 0.600 and 0.007
for vehicle and rapamycin treatment, respectively, paired
t-test; Fig. 2B), Tsc2+/− mice (p = 0.513 and 0.008 for vehicle and rapamycin treatment, respectively, paired t-test;
Fig. 2B), and TscD+/− mice (p = 0.749 and 0.003 for vehicle and rapamycin treatment, respectively, paired t-test;
Fig. 2B) and social preference in the three-chamberedsociability tests in Tsc2+/− mice (p = 0.106 and 0.007 forvehicle and rapamycin treatment, respectively, paired
t-test; Fig. 2C) and TscD+/− mice (p = 0.314 and 0.042
for vehicle and rapamycin treatment, respectively, paired

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t-test; Fig. 2C), which were similar to Tsc2+/− mice but not
Tsc1+/− mice.
Changes in gene expression in ­Tsc1+/−, ­Tsc2+/−,and ­TscD+/− mice

To evaluate similarities and differences in autistic-likebehaviors in mutant mice, gene expression levels were
profiled in the brain in mutant mice. After excluding control and low-intensity transcripts from a total of 56,745
transcripts that were included in the microarray, 45,313
transcripts were analyzed. The standard deviations of ­log2(fold-change) were 0.156, 0.165, and 0.226 for Tsc1+/−,
Tsc2+/−, and TscD+/− mice, respectively, compared with
WT mice, and the approximate curve showed a larger
variation of l­og2 (fold-change) in TscD+/− mice than in
Tsc1+/− and Tsc2+/− mice (Fig. 3A).
In Tsc1+/−, Tsc2+/−, and TscD+/− mice compared with
WT mice, 1523 transcripts, 1225 transcripts, and 4954
transcripts, respectively, were identified as DETs (twoway ANOVA, p < 0.05; Fig. 3B). To minimize false-positive changes in gene expression, only transcripts thatwere in common with at least two DETs (yellow area)
were the focus of the subsequent analysis. Of the 1119
transcripts that were obtained after excluding transcriptsthat were regulated adversely between DETs, 289 and830 were expressed upward and downward, respectively.
Hierarchical clustering analysis showed that the expression patterns of these significant transcripts in TscD+/−
mice were similar to Tsc2+/− mice but not Tsc1+/− mice
(Fig. 3C).
Among the top 20 canonical processes that were identified in Map Folders, two major categories were included:
cancer or neoplasms (e.g., lung cancer, prostatic neoplasm, hepatocellular carcinoma, and stomach neoplasm;nine processes) and diseases or pathways related toimmunity or inflammation (e.g., systemic lupus erythematosus, allergic dermatitis, and asthma; eight processes;
Fig. 3D). This result was consistent with the Tsc1 andTsc2 gene functions of suppressing tumors and regulating immune and inflammatory responses via the mTOR
pathway. Among GO process networks, the top threeenrichment processes included “cellular component
organization or biogenesis,” “regulation of cation channel activity,” and “nervous system development” (Additional file 3: Table S1). Furthermore, the 148 transcriptsthat overlapped between all three DETs were enriched in
the seven significant networks in the canonical pathway(See figure on next page.)
Fig. 2 Effects of rapamycin on social behaviors in Tsc1+/−, Tsc2+/−, and TscD+/− mice. A–C Effects of rapamycin and vehicle on the time spent in
active interaction in the social interaction test (A), time spent exploring a familiar mouse (S1) and stranger mouse (S2) in the social novelty sessionof the three-chambered sociability test (B), and time spent exploring a stranger mouse (S3) and cagemate (C) in the social preference session of thethree-chambered sociability test (C). *p < 0.05; **p < 0.01; ***p < 0.001, unpaired t-test (A) and paired t-test (B, C). Each bar indicates the mean ± SEM.
Veh vehicle, Rap rapamycin

Kashii et al. Human Genomics(2023) 17:4

Fig. 2 (See legend on previous page.)

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Kashii et al. Human Genomics(2023) 17:4

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Fig. 3 Gene expression profiles in the brain in Tsc1+/−, Tsc2+/−, and TscD+/− mice. A Histograms of the ­log2 (fold-change) (n = 45,313, binwidth = 0.02) for each mutant mouse compared with WT (cyan: Tsc1+/− vs. WT, magenta: Tsc2+/− vs. WT, blue: TscD+/− vs. WT). Solid lines represent
approximate normal distribution curves. SD standard deviation. B Venn diagram of three DETs between mutant and WT mice. Among overlappingtranscripts (yellow area), transcripts that changed in different directions (▲: upregulated, ▼: downregulated) were excluded from each entity forfurther analysis. C Heat map of valid 1119 transcript profiles in vehicle-treated samples for four genotypes (n = 4 for each genotype) that were incommon with at least two DETs and met the criteria. Hierarchical clustering was based on the relative expression value after baseline correction to
the median of vehicle-treated WT samples (red: upregulated, blue: downregulated). D Top 20 rank map folders enriched for differentially expressed
1119 transcripts in mutant mice. The blue and red folders indicate cancer/neoplasm and immunity/inflammation, respectively. FDR false discovery
rate

modeling analysis in MetaCore (p < 0.05, zScore > 100;Additional file 4: Table S2).
Differentially expressed transcripts that were reversedby rapamycin treatment in the “STAT3, IRF1, IRF4, IL‑2R
alpha chain, IFN‑γ” pathway initiated from PDLIM2

The effect of rapamycin on transcriptional changes wasinvestigated, and we assessed whether the effects corresponded to the results of the behavioral tests in mutant
mice. Among the DETs that were obtained to compare
mutant and WT mice (1523 transcripts in Tsc1+/− vs.
WT, 1225 transcripts in Tsc2+/− vs. WT, and 4954 transcripts in TscD+/− vs. WT), the expression of 180, 79,
and 174 transcripts was significantly reversed by rapamycin treatment in Tsc1+/−, Tsc2+/−, and TscD+/− mice,

respectively (two-way ANOVA, p < 0.05; Fig. 4A). The
pathway enrichment analysis was performed with canonical pathway modeling in MetaCore to elucidate signaltransduction pathways that are related to DETs between
mutant and WT mice and are reversed by rapamycin.
These DETs were enriched in seven networks, five networks, and six networks in Tsc1+/−, Tsc2+/−, and TscD+/−
mice, respectively, and “signal transducer and activatorof transcription 3 (STAT3), interferon regulatory factor 1 (IRF1), IRF4, interleukin-2R (IL-2R) α chain, andinterferon-γ (IFN-γ)” was the common network in all
mutant mice (Fig. 4A). Most DETs in the network wereregulated by STAT3, which was downstream of IRF1,
IRF4, IL-2R α chain, and IFN-γ, as well as STAT4 (Fig. 5).
The network was initiated from PDZ and LIM domainKashii et al. Human Genomics(2023) 17:4

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Fig. 4 Rapamycin-induced changes in gene expression in the brain in Tsc1+/−, Tsc2+/−, and TscD+/− mice. A The cyan-colored circle indicatestranscripts whose expression significantly changed in Tsc1+/− mice and was reversed by treatment with rapamycin ([Tsc1+/− vs. WT] ∩ [Tsc1+/−
Veh vs. Rap]). Magenta- and blue-colored circles indicate gene expression changes in Tsc2+/− and TscD+/− mice, respectively. Canonical pathway
modeling analysis for these three groups of transcripts identified seven, five, and six significant networks in Tsc1+/−, Tsc2+/−, and TscD+/− mice,
respectively (p < 0.05, zScore > 100). B Box-and-whisker plot of normalized ­log2 signal intensity for the gene expression of Pdlim2 in vehicle- or
rapamycin-treated mutant and WT mice. **p < 0.01; ***p < 0.001 (two-way ANOVA). Veh vehicle, Rap rapamycin

protein 2 (PDLIM2), which was the only gene that overlapped among DETs in all mutant mice. The gene expression of Pdlim2 was higher in mutant mice than in WT
mice and reduced by rapamycin treatment (Fig. 4B).

Discussion
Autism spectrum disorder is a polygenic and genetically heterogeneous disorder for which many genes andgenetic loci have been identified as causal factors inhuman genetic studies. The contribution of each gene or
genetic locus to ASD is low (up to a few %), indicatingthat no genetic model covers all or most all ASD cases.
The mTOR signaling pathway is a convergent pathway that is disturbed by genetic variations in the TSC1
and TSC2 (TSC), PTEN (Bannayan-Riley-Ruvalcaba
syndrome, Lhermite-Duclos syndrome, and Cowden
syndrome), FMRP (fragile X syndrome), and NF1 (neurofibromatosis type 1) genes. All of these disorders
are associated with ASD, which together account for

approximately 3–5% of overall ASD cases [12]. Thus,
Tsc1/2 heterogeneous knockout mice are considered
models for at least 3–5% of ASD cases.
Tuberous sclerosis complex manifests with such neurological symptoms as epilepsy, intellectual disability,
and syndromic ASD, the symptoms of which do not differ from non-syndromic ASD [37]. Therefore, TSC isa disease model of the pathogenesis of ASD. Tuberous
sclerosis complex is considered to be caused by pathogenic germline mutations (e.g., single nucleotide variations, deletions, insertions, and copy number variations)in either the TSC1 or TSC2 gene [38–40]. These mutations were reported to be mostly structural mutations
that induce missense (7–33%) and frameshift (24–29%)
mutations and affect splice sites (13–40%), whereas
18–28% are nonsense mutations. A total of 9–15% of
TSC patients were found to have no identifiable mutation, and genotype–phenotype causal relationships have
not yet been elucidated [39–41]. Furthermore, somaticKashii et al. Human Genomics(2023) 17:4

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Fig. 5 Signaling pathway associated with DETs in Tsc1+/−, Tsc2+/−, and TscD+/− mice with rapamycin treatment. Arrows indicate directional edges,
with main actions shown in green. All pathways are commonly initiated from PDLIM2 to STAT3 via STAT4, IRF1, IFNγ, and IRF in Tsc1+/−, Tsc2+/−, andTscD+/− mice (symbol definitions can be found at https://​portal.​genego.​com/​legen​ds/​MetaC​oreQu​ickRe​feren​ceGui​de.​pdf; accessed July 14, 2022)

TSC1 mutations were identified in lesions from patientswith a pathogenic germline mutation of TSC2 [21, 27,28], suggesting that TSC1 and TSC2 mutations may workin concert to elicit phenotypic differences among TSC
patients. The present study was performed with TscD+/−
mice as a model of TSC patients with both TSC1 andTSC2 mutations.
Tsc1+/− mice were previously reported to exhibit lower
active interaction in the social interaction test [34, 36].
Tsc2+/− mice and Ekar (Tsc2+/−) rats were also reported
to exhibit lower active interaction in the social interaction test [36, 42, 43] and impairments in social behavior
in the social novelty session of the three-chambered test[42], but they did not exhibit abnormalities in the social
approach session of the three-chambered test [42, 44].
The present results indicated that heterozygous Tsc2
mutation-induced social impairment was slightly more
severe than the heterozygous Tsc1 mutation. TscD+/−
mice exhibited impairments in social behaviors that weresimilar to Tsc2+/− mice and were associated with themTOR pathway. Tsc1 and Tsc2 proteins form a heterodimeric complex that constitutively inactivates mTORC1
through inhibition of the small guanosine triphosphatase(GTPase) protein Rheb. Tsc2 possesses the GTPaseactivating protein (GAP) domain for Rheb in the C terminus, and complex formation with Tsc1 stabilizes Tsc2and enhances its GAP activity [16]. TscD+/− mice were
expected to exhibit additive or synergistic phenotypesof both Tsc1+/− and Tsc2+/− mice. Indeed, the numberof astrocytes in the hippocampus was reported to behigher in TscD+/− mice than in Tsc1+/− and Tsc2+/− mice[30]. Tsc1f/−; Tsc2f/−; hGFAP-Cre mice have a shorter
lifespan than mice with mutations of any of these genes
alone [31]. Additionally, glial-fibrillary acidic protein(GFAP)-positive cell-specific Tsc2 conditional knockout(Tsc2GFAP1) mice exhibited a high frequency of seizuresand shorter life span than Tsc1GFAP1 conditional knockout mice [45]. Taken together with the present results,
Tsc2 rather than Tsc1 appears to be dominant for causing
neurological symptoms, including impairments in social
behaviors that are caused by heterozygous Tsc1 and/or
Tsc2 mutations, and impairments in social behaviors in
TscD+/− mice would be partially mediated through different mechanisms from astrocytic abnormalities in an
mTOR-dependent manner.
Unlike humans, which have marked individual differences, inbred mouse strains on homogeneous genetic
backgrounds can be analyzed with much smaller sample
sizes than humans. Indeed, a microarray analysis with
6–8 mice or 57 humans reported that standard deviations of the log (sample/reference) were approximately
0.3 and 0.8, respectively [46]. The Agilent microarrayhas superior detection compared with other microarray platforms [47]. Our transcriptome data that werebased on Agilent microarrays showed that standard
deviations of log (mutant/WT) in Tsc1+/−, Tsc2+/−, andKashii et al. Human Genomics(2023) 17:4

TscD+/− mice were as low as 0.158, 0.165, and 0.226,
respectively (Fig. 3A). Therefore, because the mice thatwere used in the present study were on a homogeneous genetic background and the standard deviation foreach mutant group was low, the transcriptome analysis in which four mice were analyzed for each genotype
and treatment group was considered to have the necessary power to detect differences between groups. Brainstructures are left–right asymmetrical and have different functions that are controlled by each hemisphere[48], and these asymmetries of brain structures andfunctions have been reported to be broken in ASD [49–51]. Left hemisphere impairments and microstructuralabnormalities were found in high-functioning ASD [52,53], whereas the right hemisphere failed to respond to
temporal novelty in ASD [54]. Social deficits in ASD
have been reported to be associated with gamma-band
electroencephalographic asymmetry in the right hemisphere [55], and social processing is right hemispheredominant in vertebrates [56, 57]. Unknown are theprecise brain regions and cellular subtypes that are
responsible for social deficits in ASD that are associated with TSC. Therefore, whole transcriptome analysis
was performed using the right brain hemisphere rather
than the left hemisphere and specific brain regions in
the present study.
Over 1,000 genes with alterations of gene expressions
in each mutant mice compared with WT mice were identified in the study. The magnitude of changes in gene
expression in TscD+/− mice was higher than in Tsc1+/−and Tsc2+/− mice. The number of transcripts whose
expression was significantly different from WT mice wasalso higher in TscD+/− mice than in Tsc1+/− and Tsc2+/−
mice. Our results indicated that unlike social behaviors,
gene expression in the brain in TscD+/− mice was affectedby synergistic effects of heterozygous Tsc1 and Tsc2
mutations. In the present study, few genes in the mTOR1
signaling pathway had alterations of expression in addition to the Tsc1 and Tsc2 genes. Similarly, genes in themTOR signaling pathway were not enriched in the gene
expression analysis of cortical tubers from TSC patients,suggesting that transcription does not necessarily feedback from changes in translation, whereas mTOR1
signaling regulates translation via ribosomal protein S6
kinase, polypeptide 1 (S6K1), and eukaryotic translation
initiation factor 4E binding protein 1 (4E-BP1) [21, 58].
Transgenic mice in which eukaryotic translation initiation factor 4E (Eif4e) gene expression was driven by the
β-actin promoter exhibited autistic-like behaviors anda ~ 30% increase in cap-dependent translation, but theenhanced translation was limited to only a part of proteins [59]. Our results indicated that all mutant mice
exhibited impairments in social behaviors despite large

Page 10 of 13

differences in the number of genes with alterations ofgene expression between mutant and WT mice, suggesting that impairments in social behaviors that were caused
by Tsc1 and Tsc2 mutations would be attributable tochanges in specific molecular pathways and not a generalincrease in transcription or translation.
The hierarchical clustering analysis indicated thatgene expression patterns in overlapping genes withalterations of expression between mutant and WT
mice were similar in Tsc2+/− and TscD+/− mice, whichcorresponded to the severity of impairments in social
behavior in mutant mice. Furthermore, genes whose
expression was different between Tsc1+/− and Tsc2+/−
mice were mostly enriched in the group of genes thatwere altered in Tsc2+/− and TscD+/− mice but not in
Tsc1+/− mice compared with WT mice, also indicating
the similarity of changes in gene expression between
Tsc2+/− and TscD+/− mice (Additional file 2: Fig. S2A).
The GO analysis of overlapping genes showed thatthey were enriched in cancer/neoplasm- and immunity/inflammation-related processes. These cancer/
neoplasm- and immunity/inflammation-related processes showed the up- and downregulation of genesthat were altered in Tsc2+/− and TscD+/− mice (Additional file 2: Fig. S2B), respectively, indicating that theseverity of symptoms that are related to cancer/neoplasm and immunity/inflammation would be different
between TSC patients with underlying TSC1 and TSC2
mutations.
STAT3 is phosphorylated by mTOR1 and activated in
TSC2-deficient cells, which are inhibited by rapamycin[60–62]. The expression of inflammatory gene markers,
including IL-1β, IL-6, and Toll-like receptors, and phosphorylated STAT3 increased in brain lesions in TSC
patients [63–66]. These previous studies support ourfindings that the “STAT3, IRF1, IRF4, IL-2R α chain, IFNγ” pathway network was associated with impairments insocial behaviors in mutant mice and suggest that suppressing inflammatory responses in TSC patients may
improve social deficits. This pathway network is initiatedby Pdlim2, which is ubiquitously expressed throughout the body of mice but especially in the genitourinary,nervous, and visceral organ systems. Pdlim2 encodes an
E3 ubiquitin ligase and ubiquitinates STAT3 and STAT4
to lead to their degradation [67, 68]. Pdlim2 is reported to
be associated with tumor suppression [69–71] and tumorigenesis [72, 73], but its function in the nervous systemremains unknown.

Kashii et al. Human Genomics(2023) 17:4

Page 11 of 13

Limitations
The “STAT3, IRF1, IRF4, IL-2R α chain, IFN-γ” pathway
network initiated from PDLIM2 and STAT4 was associated with impairments in social behaviors in Tsc1+/−,
Tsc2+/−, and TscD+/− mice, but the regulatory mechanisms of the mTOR pathway and social behaviors by
the pathway network were not elucidated in the present
study. It is also unclear whether the pathway network
also plays a critical role in impaired social behaviors in
autism spectrum disorders not caused by Tsc1 and Tsc2
mutations.

Supplementary Information

Conclusions
In the present study, detailed analyses of social behaviors and transcriptomes in Tsc1+/−, Tsc2+/−, andTscD+/− mice revealed the genetic pathology of impairments in social behaviors. Mutations in the TSC2 geneare likely to have dominant effects on social behaviors
rather than TSC1 gene if these mutations have similarpotency. However, rapamycin has therapeutic effects on
impairments in social behaviors caused by the mutations in both genes. It is also suggested that social
behavioral abnormalities in patients with mutations inboth the TSC1 and TSC2 genes are similar to those inpatients with mutations in the TSC2 gene. The Pdlim2mediated signaling pathway is suggested to be associated with altered brain function and impaired social
behaviors due to mutations in the TSC1 and TSC2
genes. The present findings further our understandingof genotype–phenotype relationships and may contribute to the development of medications that can ameliorate social deficits in ASD.

Acknowledgements
The authors thank the members of the animal facility at the Tokyo Metropolitan Institute for Medical Science. The authors thank Michael Arends for
proofreading the manuscript.

Abbreviations
4E-BP1	4E binding protein 1
ANOVA	Analysis of variance
ASD	Autism spectrum disorder
DETs	Differentially expressed transcripts
FDR	False discovery rate
GAP	GTPase-activating protein
GFAP	Glial-fibrillary acidic protein
GO	Gene ontology
GTPase	Guanosine triphosphatase
HSD	Honestly significant difference
IFN-γ	Interferon-γ
IL-2R	Interleukin-2R
IRF1	Interferon regulatory factor 1
LOH	Loss of heterozygosity
mTORC1	Mechanistic target of rapamycin complex 1
PDLIM2	PDZ and LIM domain protein 2
S6K1	S6 kinase, polypeptide 1
STAT3	Signal transducer and activator of transcription 3
TSC	Tuberous sclerosis complex
TSC1	TSC complex subunit 1
Tsc1 Heterozygous knockout
Tsc1+/−	
Tsc2+/−	Tsc2 Heterozygous knockout
TscD+/−	
Tsc1 And Tsc2 double heterozygous mutant
WT	Wild typeThe online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s40246-​023-​00450-2.
Additional file 1. Fig. S1. Anxiety and sensory behaviors in Tsc1+/−,
Tsc2+/−, and TscD+/− mice.
Additional file 2. Fig. S2. Differences in changes in gene expression
between Tsc1+/− and Tsc2+/− mice.
Additional file 3. Table S1. GO enrichment analysis of significant DETs in
the brain in Tsc1+/−, Tsc2+/−, and TscD+/− mice.
Additional file 4. Table S2. Canonical model pathways commonly
enriched in the brain in Tsc1+/−, Tsc2+/−, and TscD+/− mice.

Author contributions
HK and SK were involved in the gene expression analysis, gene ontology
and pathway analyses, data interpretation, and manuscript drafting. AS was
involved in the behavioral analysis. YH was involved in the preparation of mice.
YN was involved in the microarray procedure. TK and OH provided heterozygous knockout mice. MM and KI conceived the study. All authors contributed
to the manuscript and approved the submitted version.
Funding
This research was supported by Grants-in-Aid for Scientific Research from
MEXT/JSPS 24659490, 26670491, 26860836, 15H01303, 16K15565, 16K08924,
JP22H04922 (AdAMS), and AMED JP16ek0109015.
Availability of data and materials
The datasets used and/or analyzed during the current study are available fromthe corresponding author on reasonable request.

Declarations
Ethics approval and consent to participate
All animal procedures were approved by the Institutional Animal Care and Use
Committee of the Tokyo Metropolitan Institute of Medical Science (ApprovalNo. for 5 years: 18028, 19026, 20-019, 21-071, and 22-012).
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Addictive Substance Project, Tokyo Metropolitan Institute of Medical
Science, 2‑1‑6 Kamikitazawa, Setagaya‑Ku, Tokyo 156‑8506, Japan. 2 Department of Neuropediatrics, Tokyo Metropolitan Neurological Hospital, 2‑6‑1
Musashidai, Fuchu, Tokyo 183‑0042, Japan. 3 Department of Pediatrics, The
University of Tokyo Hospital, 7‑3‑1 Hongo, Bunkyo‑Ku, Tokyo 113‑8655, Japan.
4Center for Basic Technology Research, Tokyo Metropolitan Institute of Medical
Science, 2‑1‑6 Kamikitazawa, Setagaya‑Ku, Tokyo 156‑8506, Japan. 5 Department of Pathology and Oncology, Juntendo University School of Medicine,
2‑1‑1 Hongo, Bunkyo‑Ku, Tokyo 113‑8421, Japan. 6 Department of Pediatrics,
National Rehabilitation Center for Children with Disabilities, 1‑1‑10 Komone,
Itabashi‑Ku, Tokyo 173‑0037, Japan.
Received: 29 September 2022 Accepted: 18 January 2023

Kashii et al. Human Genomics(2023) 17:4

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