# Autistic-relevant behavioral phenotypes of a mouse model of cyclin-dependent kinase-like 5 deficiency disorder

Nicola Mottolese, Oceane Coiffard, Celeste Ferraguto, Athanasios Manolis, Elisabetta Ciani, Susanna Pietropaolo

► To cite this version:

Nicola Mottolese, Oceane Coiffard, Celeste Ferraguto, Athanasios Manolis, Elisabetta Ciani, et al. Autistic-relevant behavioral phenotypes of a mouse model of cyclin-dependent kinase-like 5 deficiency disorder. Autism Research, 2024, 17 (9), pp.1742-1759. 10.1002/aur.3226. hal-04764958

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RESEARCH ARTICLE

# Autistic-relevant behavioral phenotypes of a mouse model of cyclin-dependent kinase-like 5 deficiency disorder

Nicola Mottolese<fnref n="1" />,<fnref n="2" /> | Elisabetta Ciani<fnref n="1" />

Oceane Coiffard<fnref n="2" /> | Susanna Pietropaolo<fnref n="2" /> ID

Celeste Ferraguto<fnref n="2" />

Athanasios Manolis<fnref n="2" />

**Correspondence**

Susanna Pietropaolo, Institut de Neurosciences Cognitives et Intégratives d'Aquitaine, CNRS UMR 5287, Bâtiment Bordeaux Biologie Santé, 2 rue Docteur Hoffmann Martinot, 33000 Bordeaux, France.

Email: susanna.pietropaolo@u-bordeaux.fr

**Funding information**

Université de Bordeaux; Centre National de la Recherche Scientifique; Fondation Pour l'Audition; Federation Williams France; Association Autour de Williams

**Abstract**

Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is a neurodevelopmental disease caused by mutations in the X-linked CDKL5 gene and characterized by early-onset epilepsy, intellectual disability, and autistic features. To date, the etiological mechanisms underlying CDD are largely unknown and no effective therapies are available. The *Cdkl5* knock-out (KO) mouse has been broadly employed in preclinical studies on CDD; *Cdkl5*-KO mice display neurobehavioral abnormalities recapitulating most CDD symptoms, including alterations in motor, sensory, cognitive, and social abilities. However, most available preclinical studies have been carried out on adult *Cdkl5*-KO mice, so little is known about the phenotypic characteristics of this model earlier during development. Furthermore, major autistic-relevant phenotypes, for example, social and communication deficits, have been poorly investigated and mostly in male mutants. Here, we assessed the autistic-relevant behavioral phenotypes of *Cdkl5*-KO mice during the first three post-natal weeks and in adulthood. Males and females were tested, the latter including both heterozygous and homozygous mutants. *Cdkl5* mutant pups showed qualitative and quantitative alterations in ultrasonic communication, detected first at 2 weeks of age and confirmed later in adulthood. Increased levels of anxiety-like behaviors were observed in mutants at 3 weeks and in adulthood, when stereotypies, reduced social interaction and memory deficits were also observed. These behavioral effects of the mutation were evident in both sexes, being more marked and varied in homozygous than heterozygous females. These findings provide novel evidence for the autistic-relevant behavioral profile of the *Cdkl5* mouse model, thus supporting its use in future preclinical studies investigating CDD pathology and autism spectrum disorders.

**Lay Summary**

Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is a rare neurodevelopmental disease characterized by several symptoms, including autistic features. Here we demonstrate the presence of a variety of autistic-relevant behavioral alterations in the preclinical model of CDD, i.e., the *Cdkl5* knock-out (*Cdkl5*-KO) mouse, taking into account sex and age differences. Our results support the validity of this genetic mouse line for modeling CDD and its use for future preclinical pharmacological and molecular studies on this disorder.

**KEYWORDS**

acoustic startle, CDD, mouse pups, neurodevelopmental disorders, social behaviors, social interaction, ultrasonic vocalizations

<footnote n="1">¹Department of Biomedical and Neuromotor Sciences, University of Bologna, Italy</footnote>

<footnote n="2">²CNRS, EPHE, INCIA, Univ. Bordeaux, Bordeaux, France</footnote>
# INTRODUCTION

Mutations in the X-linked CDKL5 gene cause CDKL5 deficiency disorder (CDD), a rare neurodevelopmental disease (NDD), affecting up to 1:40,000 live births (Fehr et al., 2013). The CDKL5 gene has a coding region which is mainly orthologous and well-conserved between human and mouse (Hector et al., 2016); it codes for a serine/threonine kinase that is highly expressed in the central nervous system of both mammalian species (Hector et al., 2016). This gene has both a cytosolic and nuclear localization, which varies across brain areas and during development (Rusconi et al., 2008); it is present in postsynaptic structures, where it can regulate dendritic spine maturation and density, and modulate excitatory synaptic function (Della Sala et al., 2016; Ricciardi et al., 2012). In mice, Cdkl5 is strongly upregulated postnatally with high levels of expression in the forebrain (Hector et al., 2016; Rusconi et al., 2008).

CDD is characterized by early-onset epilepsy, generalized hypotonia, intellectual, motor, and visual disabilities (Fehr et al., 2013; Jakimiec et al., 2020; Leonard et al., 2022; Olson et al., 2019). All individuals diagnosed with CDD also display features commonly associated with autism spectrum disorder (ASD), including social avoidance, communication deficits, as well as stereotypic behaviors (Fehr, Downs, et al., 2016; Fehr, Wong, et al., 2016). Therapeutic approaches to treat the whole spectrum of CDD symptoms are lacking (Olson et al., 2021); pharmacological interventions are mostly limited to selected neurologic features of the disorders, with a major focus on epilepsy management (Hong et al., 2022); the latter is a highly challenging task, because of the refractory nature of CDD seizures. Hence, there is an urgent need to design CDD-specific therapeutic approaches targeting the molecular pathological mechanisms of the syndrome (Van Bergen et al., 2022), and treating its multiple behavioral and neurological phenotypes.

In order to promote the identification of novel therapeutic targets and advance our understanding of the etiopathological mechanisms underlying this disorder, the Cdkl5-knock-out (KO) mouse models of CDD were engineered (Amendola et al., 2014; Okuda et al., 2017; Wang et al., 2012). Cdkl5-KO mice recapitulate different features of CDD, including cognitive and visual impairments, compromised motor coordination and locomotion (Amendola et al., 2014; Della Sala et al., 2016; Fuchs et al., 2014; Fuchs, Gennaccaro, et al., 2018; Gennaccaro et al., 2021; Lo Martire et al., 2017; Trazzi et al., 2016; Wang et al., 2012). Reduced social interest, stereotyped movements, and social communication deficits were also described in Cdkl5-KO mice, although without an extensive characterization of the social phenotypes, thus suggesting the presence of autistic-relevant phenotypes in this mouse model (Amendola et al., 2014; Fuchs,

Gennaccaro, et al., 2018; Gao et al., 2020; Jhang et al., 2017; Okuda et al., 2017; Wang et al., 2012).

Despite the large body of evidence describing the behavioral CDD-like phenotypes of Cdkl5-KO mice, the majority of existing preclinical studies have focused so far on male mutant mice. Cdkl5 mutant females have been poorly studied and their autistic-relevant behavioral phenotypes have not been extensively characterized. This lacuna is surprising, since CDD primarily affects girls who are heterozygous for CDKL5 deficiency, with a female to male ratio of 4:1 (Lopez-Rivera et al., 2020; Symonds et al., 2019). Furthermore, although male patients are generally thought to develop more severe symptoms than females (Fehr et al., 2015; Fehr, Downs, et al., 2016), recent results suggest otherwise (Demarest, Olson, et al., 2019; Demarest, Pestana-Knight, et al., 2019): it is indeed possible that the severity of pathological phenotypes has been underestimated in female CDD patients, masked by the higher symptomatic variability due to the X chromosome random inactivation (Demarest, Olson, et al., 2019; Demarest, Pestana-Knight, et al., 2019). Furthermore, most behavioral studies in the Cdkl5-KO mouse model have been performed in adult mice, so little is known about the severity of the behavioral phenotypes of Cdkl5-KO mice during early life phases (Jhang et al., 2017; Tassinari et al., 2023).

In contrast, the early brain phenotypes of Cdkl5-KO mice have already been characterized. Several alterations have been reported in the brains of developing and adolescent Cdkl5-KO mice, including impaired dendritic arborization of cortical and hippocampal pyramidal neurons, reduced spine density and maturation, increased rate of apoptotic cell death of post-mitotic granule precursors in the dentate gyrus of the hippocampus, and microglia over-activation (Della Sala et al., 2016; Tassinari et al., 2023). In addition, functional magnetic resonance imaging (fMRI) and electrophysiological analyses revealed altered excitatory connectivity, transmission, and plasticity in the brains of Cdkl5-KO pups and juveniles (Della Sala et al., 2016; Tassinari et al., 2023). These findings support a critical role of the Cdkl5 gene in the maintenance of the brain excitation/inhibition (E/I) imbalance. An E/I imbalance in the neural circuit may be indeed at the root of the early seizure phenotype induced by Cdkl5 mutations in CDD patients and mouse models, and could be also involved in their autistic-relevant phenotypes (Bruining et al., 2020). The latter may also be linked to the disrupted expression of several genes involved in ASD, which has been described in specific brain regions of young Cdkl5-KO mutants (Jhang et al., 2017). Hence, longitudinal studies determining the post-natal stage at which CDD-like behavioral alterations emerge in Cdkl5-KO female and male mice, and the evolution of these abnormalities into adulthood, are needed, since they are of crucial relevance to advance preclinical studies on a neurodevelopmental disorder like CDD.
Here we longitudinally assessed autistic-relevant behavioral phenotypes in *Cdkl5* mutant mice of both sexes, including the first three post-natal weeks as well as adulthood. Physical and behavioral development was evaluated in mouse pups on post-natal days (PNDs) 7, 14, and 21 through the assessment of body weight/length, ultrasonic vocalizations (USVs), and exploration in the open field. Ultrasonic communication, activity and anxiety-like behaviors in the open field were also assessed in adulthood, that is, at 7 months of age, together with additional ASD-relevant domains including stereotypic clasping, social interaction and cognitive abilities in the context freezing assay. Furthermore, sensory-motor response in the acoustic startle test was evaluated, since hyperacusis to low-intensity stimuli is an ASD-relevant phenotype which is often described in mouse models of ASD (e.g., (Ferraguto et al., 2023; Michalon et al., 2012; Zhang et al., 2014)). Males and females were tested, including both heterozygous and homozygous mutations for the latter, thus allowing to assess potential X-linked gene-dose behavioral effects in female mutants.

# MATERIALS AND METHODS

## Ethics approval

All experimental procedures were in accordance with the European Communities Council Directive 2010/63/EEC, and approved by local ethical committee (“Comité d’Ethique pour l’experimentation animale de Bordeaux”, CE 50) and the French Ministry (“Ministère de l’enseignement supérieur de la recherche et de l’innovation”).

## Animals

Subjects were *Cdkl5* mutant mice and their wild-type littermates (maintained on B6 background (Amendola et al., 2014)). A schematic representation of the

experimental design is provided in Figure 1. A cohort of 27 male (16 +/Y, 11 −/Y) and 34 female (7 +/+, 15 +/−, 12 −/−) mice was behaviorally assessed during early post-natal development (i.e., at PNDs 7, 14, and 21); a sub-cohort of the same batch of animals (including 12 +/Y and 6 −/Y males, 7 +/+, 11 +/− and 10 −/− females) was then tested at adulthood (i.e., at 7 months of age). Hind-limb clasping was evaluated in an independent batch of 22 male (12 +/Y, 10 −/Y) and 33 female (11 +/+, 11 +/−, 11 −/−) 7-month-old mice; clasping is often considered as an index of stereotypic behavior (Tassinari et al., 2022), since it consists of an inappropriate sequence of body postures lacking any goal or function (Garner, 2005). The 7-month-old age was chosen to test adult mice, since this age corresponds to full maturity in B6 mice (Flurkey et al., 2007), and it allowed to maximize the time interval occurring from previous behavioral testing at infancy, concomitantly avoiding the middle age (i.e., starting at 10 months (Flurkey et al., 2007)). Indeed, 6–7-month-old mice are not uncommonly used in research on mouse models of NDDs and ASD (e.g., (Kat et al., 2022; Lemaire-Mayo et al., 2020; Piquemal et al., 2020)) and they are often employed as the “young” control group in preclinical studies on aging and Alzheimer disease (e.g., (Bach et al., 1999; Pietropaolo et al., 2008; Radulescu et al., 2021; Webster et al., 2014)).

Mice were originally obtained from the laboratory of Prof. Ciani (University of Bologna, Italy) and bred in the animal facility of the University of Bordeaux for two generations. Breeding trios were formed by mating two *Cdkl5* heterozygous females with a WT C57BL/6J (B6) male purchased from Janvier Labs (Le Genest St Isle, France) or with a *Cdkl5* hemizygous male bred at the Bordeaux animal facility. On PND 3 pups were marked by paw tattoo, using a non-toxic odor-less ink (Ketchum permanent Tattoo Inks green paste; Ketchum MFG. Co, Lake Luzerne, NY, USA), and tail samples were collected for DNA extraction and subsequent PCR genotype assessment as previously described (Amendola et al., 2014).

<figure><img src="image_2.png" /><figcaption>**FIGURE 1** Experimental plan of the study. A cohort consisting of 27 male (16 +/Y, 11 −/Y) and 34 female (7 +/+, 15 +/−, 12 −/−) mice was used for Study 1, while a sub-cohort of the same batch of animals was subjected to Study 2; the latter included 18 males (12 +/Y, 6 −/Y) and 28 females (7 +/+, 11 +/−, 10 −/−). Body weight was also assessed at adulthood, that is, 24 h before the beginning of behavioral tests. Hind-limb clasping, that is, an index of stereotypic behavior (Tassinari et al., 2022), was evaluated in an independent batch of 7-month-old male (12 +/Y, 10 −/Y) and female (11 +/+, 11 +/−, 11 −/−) mice. ASR, acoustic startle response; BL, body length; BW, body weight; EXP, exploration; FCND, fear conditioning; OF, open field; PND, post-natal day; SI, social interaction; TD, testing day; USVs, ultrasonic vocalizations.</figcaption></figure>
Animals were weaned on PND 22 and housed in same-sex cages in groups of 3–5 animals/cage in polycarbonate standard cages (33 × 15 × 14 cm in size; Tecnoplast, Limonest, France) with cotton nestlets as cage enrichment and kept in an air-conditioned room (temperature 21 ± 1°C; humidity 55%) with lights on from 07:00 a.m. to 07:00 p.m. All behavioral tests were carried out during the light phase of the cycle (between 9 a.m. and 4 p.m.) by an experimenter who was blind to the genotype of the subjects. Mice were habituated to the experimental room for at least 30 min before the beginning of each behavioral test.

A detailed description of the methods used for pup and adult behavioral assessment is provided in Data S1.

## Statistical analysis

Statistical analyses were performed separately in the two sexes, since the X-linked mutation was differently expressed in male (hemizygous) and female (heterozygous or homozygous) mutants. Body weight and length data were analyzed by an ANOVA with genotype as the between-subject factor and testing days as the within-subject factor. Data from pups' behavior (exploration and USVs) were instead separately analyzed on PND 7, 14 and 21 since the testing conditions (i.e., size of the testing apparatus) changed across

testing days, in order to adapt to the physical growth of the pups. Behavioral data from adult mice were analyzed by an ANOVA with genotype as the between-subject factor and additional within-subject factors depending on the test (e.g., time-bins and startle intensities). Post-hoc comparisons were performed using Tukey's-Kramer test.

All analyses were conducted using the software Statview and SPSS and α was set at 0.05. The exact number of mice is indicated in the legend of each figure; slight differences across tests may exist due to technical reasons (e.g., loss of video or audio recordings).

## RESULTS

### Physical assessment of mouse pups

As expected, body weight increased from PND 7 to PND 21 in mice of both sexes [day effect in males: $F(2,50) = 798.56, p < 0.0001$; in females: $F(2,62) = 977.36, p < 0.0001$; Figure 2a,b]; this weight gain was less pronounced in -/Y males [genotype x day interaction: $F(2,50) = 4.38, p = 0.02$; Figure 2a] and -/- females [genotype x day interaction: $F(4,62) = 5.21, p = 0.001$; Figure 2b]. Body length also increased with time [in males: $F(2,50) = 527.80, p < 0.0001$; in females: $F(2,62) = 360.09, p < 0.0001$; Figure 2c,d], but no

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<figure><img src="image_4.png" /></figure>

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<figure><img src="image_6.png" /></figure>

**FIGURE 2** Body growth of Cdk15 pups. Body weight (a, b) and length (c, d) of Cdk15 males (n = 16 and 11 for +/Y and -/Y mice) and females (n = 7, 15, and 12 for +/+, +/-, and -/- mice). Data are expressed as mean ± standard error of the mean (SEM). PND, post-natal day. \* *p* < 0.05.
difference was observed between genotypes [genotype effect and its interaction with day, all ns].

## Behavioral assessment of mouse pups

### Ultrasonic vocalizations

Quantitative and qualitative alterations in USVs were detected in male and female mutant pups, although their expression across days slightly differed between the two sexes. Mutant -/Y males emitted less and shorter USVs than their WT littermates on PND 14 [genotype effect on USV number and duration in males: F(1,23) = 5.51 and 6.91, *p* = 0.03 and 0.01; Figure 3a,c]. The USVs uttered by -/Y male pups on PND 14 were also characterized by

a lower mean frequency, an abnormality that was already evident on PND 7 [genotype effect in males on PND 7 and 14: F(1,23) = 4.70 and 10.23, *p* = 0.04 and *p* = 0.004, respectively; Figure 3e]. Similarly to males, mutant +/− and -/− females also emitted less USVs than WT mice on PND 14 [genotype effect on this day: F (2,31) = 4.02, *p* = 0.03; Figure 3b], but this deficit persisted on PND 21 in -/− mutants [genotype effect: F (2,31) = 3.49, *p* = 0.04; Figure 3b]; in contrast to males, USV mean duration was unaltered in mutant females at all tested days [genotype effect on all PNDs, ns; Figure 3d]. As observed in males, the USVs uttered by -/− mutant female pups were characterized by a lower mean frequency, but this anomaly was detected only on PND 14 [genotype effect in females on PND 14: F(2,31) = 3.71, *p* = 0.04; Figure 3f].

<figure><img src="image_7.png" /><figcaption>**FIGURE 3** Characterization of main spectrographic parameters of ultrasonic vocalizations (USVs) of *Cdkl5* pups. Number (a, b), mean duration (c, d) and mean frequency (e, f) of the USVs emitted by *Cdkl5* mutant pups and their WT littermates during a 3-min isolation session. *Cdkl5* males (15 +/Y and 10 −/Y) and females (7 +/+, 15 +/− and 12 −/−) were repeatedly tested at post-natal day (PND) 7, 14, and 21. Data are expressed as mean ± SEM. \* *p* < 0.05.</figcaption></figure>
Additional and more subtle qualitative abnormalities were detected in the USVs emitted by mutant *Cdkl5* mice. These alterations were detected in both male and female mutants, but once again, their time-dependent expression differed between sexes. Male -/Y mice emitted on PND 14 more “short” and less “down” calls than their +/Y littermates [genotype effect on percent of these two call types on this day:

genotype effect: $F(1,23) = 5.21$ and $5.37$, $p = 0.03$; Figure 4b]. Similarly, female -/- mutants uttered less “down” calls on PND 14 compared to their +/+ littermates [genotype effect: $F(2,31) = 4.33$, $p = 0.02$; Figure 4e]. In addition, -/- females, as well as +/- mutants, emitted also more “flat” USVs than their WT littermates on PND 7 [genotype effect: $F(2,31) = 4.32$, $p = 0.02$; Figure 4d].

<figure><img src="image_8.png" /></figure>

**FIGURE 4** Qualitative categories of ultrasonic vocalizations (USVs) of *Cdkl5* pups. Call types were automatically categorized (see also Figure S1) and the relative percent composition of male and female USVs was separately analyzed on post-natal day (PND) 7 (a and d), 14 (b and e) and 21 (c and f). Calls were obtained from a total of 25 males (15 +/Y and 10 -/Y) and 34 females (7 +/+, 15 +/- and 12 -/-), as described for Figure 3. Detached slices indicate call categories where significant genotype differences emerged. *$p < 0.05$ versus same-sex WT (+/Y males or +/+ females) on the same PND.
## Locomotion and exploration

Cdkl5 mutation did not affect locomotion of either male or female pups on any of the testing days [genotype effect on PND 7, 14 and 21 in males or females, ns; Figure 5a,c]. On PND 21 mutant Cdkl5 mice of both sexes displayed higher levels of anxiety-like behavior than their WT littermates, spending significantly less time in the central area of the open field [genotype effect in males and females, respectively: $F(1,25) = 13.91, p = 0.001$; $F(2,31) = 29.11, p < 0.0001$; Figure 5b,d]. In females, this effect was observed in both +/− and −/− mutants in comparison to +/+ littermates (Figure 5d).

## Behavioral assessment of adult mice

Twenty-four hours before the beginning of behavioral testing, the body weight of all mice was assessed; no genotype effect was detected either in male or female mice with the following mean ± SEM values (expressed in gr): $36.6 \pm 2.41$ and $37.19 \pm 2.04$ for +/Y and −/Y

males; $27.74 \pm 1.77$, $27.12 \pm 0.64$, and $28.16 \pm 1.01$ for +/+, +/−, and −/− females.

## Hind-limb clasping

Cdkl5 mutant mice spent more time performing the stereotypic clasping behavior than their WT littermates and this genotype effect was evident in both males [$F(1,20) = 41.35, p < 0.0001$; mean ± SEM (sec) in +/Y: $0.26 \pm 0.12$, in −/Y: $2.97 \pm 0.44$] and females [$F(2,29) = 10.74, p = 0.0003$; mean ± SEM (sec) in +/+: $0.08 \pm 0.05$, in +/−: $3.72 \pm 0.67$ and −/−: $3.48 \pm 0.75$], the latter showing the effects of the mutation both in +/− and −/− mice.

## Locomotion and exploration in the open field

At adulthood Cdkl5 mutation affected locomotion and anxiety-like behavior in both sexes. −/Y males were overall more active than their +/Y littermates [genotype effect

**FIGURE 5** Locomotion and exploration in Cdkl5 pups. Explorative behaviors of mouse pups were assessed during the 3-min isolation sessions employed for USV recording. Cdkl5 males (16 +/Y and 11 −/Y) and females (7 +/+, 15 +/− and 12 −/−) were assessed at post-natal day (PND) 7, 14, and 21. Mice were tested on the different PNDs in a plastic container with dimensions that increased with the animals' body size (10 × 8 × 6 cm for PND 7; 8 × 8 × 10 cm for PND 14; 25 × 15 × 22 cm for PND 21). Locomotion (a and c) was scored as the distance traveled on each testing day, while anxiety-like behavior (b and d) was assessed only on PND 21 by analyzing the time spent in the central area of the testing arena. Data are expressed as mean ± SEM. \*p < 0.05.

<figure><img src="image_9.png" /></figure>
<figure><img src="image_10.png" /><figcaption>FIGURE 6 Locomotion and exploration in adult *Cdkl5* mice. Mean speed (a, e), total distance traveled (b, f), locomotor habituation (c, g) and levels of anxiety-like behavior (d, h) were evaluated during a 15-min session of the open field test. Adult (7-month-old) mice included a total of 17 males (11 +/Y and 6 −/Y) and 28 females (7 +/+, 11 +/− and 10 −/−). Data are expressed as mean ± SEM. \*p < 0.05.</figcaption></figure>

on mean speed and distance moved in males: F(1,15) = 23.56 and 7.65, *p* = 0.0002 and 0.01; Figure 6a,b), but they displayed unaltered locomotor habituation within the open field testing session [5-min bin effect: F(2,30) = 3.75, *p* = 0.03; genotype x 5-min bin interaction, ns; Figure 6c]. −/Y males also displayed higher anxiety levels, spending less time in the central area of the open field than +/Y littermates [genotype effect: F(1,15) = 8.23, *p* = 0.01; Figure 6d]. *Cdkl5* female mutants (both +/− and −/−) were also more active than their +/+ littermates, although this hyperactivity was more pronounced in −/− females [genotype effect on mean speed and distance moved: F(2,25) = 9.61 and 7.10, *p* = 0.0008 and 0.003; Figure 6e,f]. +/− and −/− females also displayed a deficit in locomotor habituation, that was once again more marked in −/− mutants [genotype x 5-min bin interaction: F(4,50) = 6.59, *p* = 0.0002; Figure 6g]. Similar to male data, *Cdkl5* mutant females showed increased anxiety-like behavior in the open field, but this phenotype was observed in −/− mice only [genotype effect: F(2,25) = 14.37, *p* < 0.0001; Figure 6h].

## Social interaction

In males, *Cdkl5* mutant mice displayed less social behaviors than their WT littermates [genotype effect: F(1,16) = 9.83, *p* = 0.01; Figure 7a], and they also emitted less USVs [genotype effect: F(1,16) = 20.35, *p* = 0.0004; Figure 7c] with reduced duration and mean frequency [genotype effect: F(1,16) = 4.93 and 71.89, *p* = 0.04 and *p* < 0.0001; Figure 7e,g]. The same pattern was observed in females, where *Cdkl5* mutation again led to decreased social behaviors in both +/− and −/− mice, this effect being more pronounced in homozygous mutants [genotype effect: F(2,25) = 7.75, *p* = 0.002; Figure 7b]. The number of USVs was significantly reduced only in −/− females, as well as their mean frequency [genotype effect: F(2,23) = 4.59 and 5.01, *p* = 0.02; Figure 7d,h], whereas USV duration was unaltered in both groups of mutant females [genotype effect, ns; Figure 7f].

As previously observed in mouse pups, subtle qualitative differences affected the USVs uttered by adult *Cdkl5* mutant mice of both sexes, although this time the
mutation induced more varied effects in males (Figure 8). Cdkl5 mutant −/Y males emitted less Up, Down, Chev-ron and U-shape USVs [genotype effect: F(1,16) = 18.32,

11.27, 8.04, and 5.16; *p* = 0.0006, 0.004, 0.01, and 0.04], but more short and flat calls [genotype effect: F(1,16) = 16.73 and 52.46; *p* = 0.0009 and *p* < 0.0001] compared to their +/Y littermates (Figure 8a,b). In females only the percentage of total complex calls was higher in −/− mutant mice compared to their WT littermates [genotype effect: F(2,23) = 3.94, *p* = 0.03; Figure 8c–e].

<figure><img src="image_11.png" /></figure>

## Acoustic startle

Acoustic startle was unaltered in Cdkl5 mutant males, displaying an increasing response with the stimulus intensity similar to their WT littermates [genotype effect, ns; stimulus intensity effect: F(3,48) = 13.23, *p* < 0.0001; Figure 9a]. In females, an enhanced acoustic startle response was instead displayed by −/− mutants, although only at the lowest stimulus intensity [genotype x stimulus intensity interaction: F(6.75) = 2.49, *p* = 0.03; Figure 9b].

## Fear conditioning

During the conditioning phase, all animals showed an increased percentage of freezing time at the end of the testing session, that is, after the last shock exposure, suggesting successful fear conditioning [shock exposure effect in males: F(1,14) = 47.77, *p* < 0.0001; in females: F(1,25) = 350.82, *p* < 0.0001; genotype effects, all n.s.; Figure 9c,d]. During the context test, Cdkl5 mutation induced memory deficits in both sexes: freezing time was significantly reduced in −/Y male mice [genotype effect: F(1,14) = 6.41, *p* = 0.02; Figure 9e] and in +/− and −/− mutant females [genotype effect: F(2,25) = 13.85, *p* < 0.0001; Figure 9f]. This effect became more evident with time in males [genotype x 2-min bin interaction: F(2,28) = 4.06, *p* = 0.03; Figure 9g] and females [genotype x 2-min bin interaction: F(4,50) = 3.62, *p* = 0.01; Figure 9h], although in the latter it emerged already at the beginning of the testing session.

**FIGURE 7** Social interaction and ultrasonic vocalizations at adulthood. The total amount of time spent in affiliative behaviors (i.e., sniffing and exploring the stimulus mouse) was assessed during a 3-min session of direct social interaction with an unfamiliar 6-month-old CD1-Swiss female stimulus mouse (a, b). Spectrographic analysis of ultrasonic vocalizations (USVs) was conducted to evaluate their number (c, d), mean duration (e, f) and mean frequency (g, h). Data from social affiliation (a, b) were collected from 18 males (12 +/Y and 6 −/Y) and 28 females (7 +/+, 11 +/−, 10 −/−) mice. All USV parameters (c–h) were measured on calls obtained from 18 male (12 +/Y and 6 −/Y) and 26 female (7 +/+, 10 +/−, 9 −/−) mice. Data are expressed as mean ± SEM. \* *p* < 0.05.
<figure><img src="image_12.png" /><figcaption>**FIG URE 8** Classification of ultrasonic vocalizations at adulthood. Call type composition of ultrasonic vocalizations emitted by 7-month-old *Cdkl5* males (12 +/Y and 6 −/Y; a and b) and females (7 +/+, 10 +/−, 9 −/−; c, d, and e). Significant differences in call categories are marked as slices detached from the remaining types. \*p* < 0.05 versus same-sex WT littermates (+/Y males or +/+ females).</figcaption></figure>

DISCUSSION

Our results, summarized in Table 1, provide an extensive
behavioral characterization of the *Cdkl5*-KO mouse
model of CDD, achieved by longitudinally testing male
and female mice during early life and in adulthood. Over-
all, our findings suggest that multiple autistic-relevant
behaviors can be recapitulated by the *Cdkl5*-KO preclini-
cal model, including communication, social, motor, emo-
tional, and cognitive abnormalities. These phenotypes
were displayed by *Cdkl5* mutants of both sexes, although
few and subtle phenotypic sex differences were detected.
Heterozygous +/− females, representing the common
condition of CDD female patients (Mangatt et al., 2016),
displayed several autistic-relevant phenotypes at early
and adult ages; these defects were less varied and pro-
nounced than those detected in −/− female mice, suggest-
ing X-linked dosage-dependent behavioral effects of
*Cdkl5* expression (Table 1).

**Age-dependent autistic-relevant phenotypes in**
**Cdkl5-KO mice**

Overall, no major abnormalities in physical development
and body weight were detected in *Cdkl5* mutant mice of
both sexes (Table 1), as previously reported (Amendola
et al., 2014; Fuchs, Gennaccaro, et al., 2018; Gennaccaro
et al., 2021; Mottolese et al., 2023; Okuda et al., 2018): in
mutant pups, body length was indeed unaltered during
the first three post-natal weeks, while their body weight
was lower only at PND 21, but not in adulthood. The
lack of body weight differences between mutant and WT
mice suggests that the reduced body weight observed in
CDD patients (Jakimiec et al., 2020) does not represent a
constitutive CDKL5-depedent clinical feature, but may
be rather linked to the gastro-intestinal (Mangatt
et al., 2016) and feeding (Frullanti et al., 2019) problems

often associated to CDD. Furthermore, the absence of
body weight alterations in mutant mice ruled out a major
confounding factor, which could have affected several
behavioral measures. Marked reductions in animals'
body weight are for instance known to lead to reduced
USV emission rates in pups and adult mice because of
the decreased pulmonary-thoracic size (de Chaumont
et al., 2021; Scattoni et al., 2008).

Locomotor activity and anxiety-like behaviors, that
is, two CDD- and ASD-relevant domains (Frullanti
et al., 2019; Persico et al., 2019), were longitudinally
assessed through the open field test both in pups and
adult mice. Our findings demonstrated that while –/Y,
+/– and –/– mice displayed hyperactivity only in adult-
hood, their levels of anxiety-like behavior were increased
compared to WT littermates already at PND 21. These
results are in line with previous data from *Cdkl5* mutant
males (Jhang et al., 2017; Mottolese et al., 2023; Okuda
et al., 2018; Trazzi et al., 2018) and females (Fuchs
et al., 2022; Fuchs, Gennaccaro, et al., 2018). Hyperac-
tivity has been described in other genetic mouse models
of ASD (Ey et al., 2018; Penagarikano et al., 2011;
Pietropaolo & Subashi, 2014), where it sometimes exerted
a confounding reducing impact on anxiety-like behaviors
(Kat et al., 2022). Here, hyperactivity was instead associ-
ated with increased levels of anxiety-like behaviors in
*Cdkl5* mutant mice, thus enhancing the ASD-related face
validity of this mouse line.

Social communication was another ASD-relevant
domain, which could be assessed both in pups and in
adult mice, although using different eliciting contexts. As
in most studies on ASD mouse models (Caruso
et al., 2020), USV emission during development was
assessed in isolated pups, while in adult male and female
mice USVs were recorded during social encounters with
an adult female intruder [see also (Premoli et al., 2022)].
USV abnormalities were mostly evident in *Cdkl5* pups on
PND 14, when reduced call number and mean frequency
<figure><img src="image_13.png" /><figcaption>**FIGURE 9** Acoustic startle response and fear conditioning at adulthood. Acoustic startle response (a, b) and fear conditioning (c to h) was assessed in 18 males (12 +/Y and 6 -/Y) and 28 females (7 +/+, 11 +/− and 10 −/−). Acoustic startle was measured through the presentation of 4 sound intensities over the 66 dB background (bkgd) of white noise; startle data were subjected to natural logarithmic (ln) transformation in order to meet the ANOVA normality requirements (a, b). To test fear conditioning, mice received on Day 1 five 2-sec electric shocks (ITI = 1 min) after 2 minutes of habituation; the session ended 1 min after the presentation of the last shock. For this conditioning phase (c, d), percent freezing was assessed during the 2-min initial habituation phase (pre-shock) and the last 1-min interval after the last shock presentation (post-shock). On Day 2 animals were put back in the same chambers for 6 minutes, without receiving any shock (e, f). For this context test, percent freezing was scored across 2-min bins (g, h). Data are expressed as mean ± SEM. \*p < 0.05.</figcaption></figure>

were observed in mutants of both sexes, while the duration was reduced in mutant males only. While these mutant phenotypes became undetectable on PND 21, the same USV alterations (concerning call number, mean frequency and duration) were confirmed in adult mutant mice (Table 1). The lack of genotype differences on USVs on PND 21 may be due to the overall call rate decrease and loss of ethological relevance of pup USVs at this age, although it could also reflect a discontinuity in the molecular alterations potentially underlying communication deficits in *Cdk15* mutant mice. The expression of *Fork-head-box P2* (*FoxP2*) gene, involved in ASD-relevant behaviors in mice (Chen et al., 2016; Enard et al., 2009), is for instance markedly disrupted in the striatal regions of the brains of *Cdk15* mutant mice, and its role in their communication deficits has been suggested (Jhang et al., 2017).

Nonetheless, it is important to underline that here USVs were analyzed as early as PND 7, in order to concomitantly assess locomotor activity and to avoid earlier interference in mother-pup interactions which may exert long-term confounding effects on the behaviors tested at adulthood in the same batch of mice. This age is not uncommonly chosen as the earliest testing point to assess pup USVs in mouse models of ASD (e.g., (Nolan et al., 2020; Oddi et al., 2015; Radulescu et al., 2021; Wohr et al., 2008)) and it corresponds to the peak in the number of USVs, thus allowing extensive qualitative and call type analyses. During the first post-natal week, mouse pups indeed increase their number of vocalizations, while a subsequent decrease follows during the second post-natal week, that is, a typical inverted U-shaped developmental pattern (Branchi et al., 2001; Sungur et al., 2016). It is therefore evident that only the descendent branch of the inverted U-shaped curve was analyzed here; future studies specifically focusing on the ascendant part of this curve, that is, assessing USVs during the first post-natal week, may provide additional interesting information about the early communicative phenotype of *Cdk15-KO* mice.

The reduced USV number observed here in *Cdk15* pup and adult mutants is in line with previous reports on this (Jhang et al., 2017) and other ASD mouse models (Ey et al., 2011; Scattoni et al., 2008; Scattoni et al., 2011; Schmeisser et al., 2012). Previous findings on the call duration and frequency in other models of ASD show instead a more inconsistent pattern; nonetheless, previous studies have suggested that these two USV parameters are highly sensitive to manipulations of the maternal environment in mouse pups (Granata et al., 2021; Oddi et al., 2015) and may positively correlate with social interest in adult mice of both sexes (de Chaumont et al., 2021). Furthermore, differences in ultrasonic call duration and frequency are supposed to represent a valuable index of changes in the emotional
<table><caption>TABLE 1 Overview of the autistic-relevant phenotypes of Cdk15-mutant mice during pre-weaning development and in adulthood.</caption><thead><tr><th rowspan="2">Domain tested</th><th rowspan="2">Variable measured</th><th rowspan="2">Figures</th><th colspan="4">PUPS</th><th colspan="2">ADULTS</th></tr><tr><th colspan="2">PND 7</th><th colspan="2">PND 14</th><th colspan="2">PND 21</th></tr><tr><th></th><th></th><th></th><th>♂</th><th>♀</th><th>♂</th><th>♀</th><th>♂</th><th>♀</th><th>♂</th><th>♀</th></tr></thead><tbody><tr><td>Physical growth</td><td>Body weight</td><td>2a,b; text</td><td>=</td><td>=</td><td>=</td><td>=</td><td>↓ in<br/>-/-Y</td><td>↓ in<br/>-/-</td><td>=</td><td>=</td></tr><tr><td>Locomotor activity</td><td>Mean speed, total distance traveled</td><td>5a,c and<br/>6a-c,<br/>e-g</td><td>=</td><td>=</td><td>=</td><td>=</td><td>=</td><td>=</td><td>↑ in -/-Y</td><td>↑ in +/-<br/>and -/-</td></tr><tr><td>Anxiety-like behavior</td><td>% Time in arena center</td><td>5b,d and<br/>6d,h</td><td>=</td><td>=</td><td>=</td><td>=</td><td>↓ in<br/>-/-Y</td><td>↓ in<br/>+/-<br/>and<br/>-/-</td><td>↓ in -/-Y</td><td>↓ in +/-<br/>and -/-</td></tr><tr><td rowspan="4">Ultrasonic communication (USVs)</td><td>Number</td><td>3a,b and<br/>7c,d</td><td>=</td><td>=</td><td>↓ in -/-Y</td><td>↓ in +/-<br/>and -/-</td><td>=</td><td>=</td><td>↓ in -/-Y</td><td>↓ in -/-</td></tr><tr><td>Mean duration</td><td>3c,d and<br/>7e,f</td><td>=</td><td>=</td><td>↓ in -/-Y</td><td>=</td><td>=</td><td>=</td><td>↓ in -/-Y</td><td>=</td></tr><tr><td>Mean frequency</td><td>3e,f and<br/>7g,h</td><td>=</td><td>=</td><td>↓ in -/-Y</td><td>↓ in -/-</td><td>=</td><td>=</td><td>↓ in -/-Y</td><td>↓ in -/-</td></tr><tr><td>% Call types</td><td>4 and 8</td><td>=</td><td>↑ flat in<br/>+/- and<br/>-/-</td><td>↓ down,<br/>↑ short,<br/>↑ flat in<br/>-/-Y</td><td>↓ down in<br/>+/- and<br/>-/-</td><td>=</td><td>=</td><td>↓ down, ↑ short, ↑ flat;<br/>↓ up, ↓ chevron,<br/>↓ U-shape in -/-Y</td><td>↑ complex<br/>in -/-</td></tr><tr><td>Stereotypic behaviors</td><td>Time in hind-limb clasping</td><td>text</td><td></td><td></td><td></td><td></td><td></td><td></td><td>↓ in -/-Y</td><td>↓ in +/-<br/>and -/-</td></tr><tr><td>Social interaction</td><td>Time in affiliative behaviors</td><td>7a,b</td><td></td><td></td><td></td><td></td><td></td><td></td><td>↓ in -/-Y</td><td>↓ in +/-<br/>and -/-</td></tr><tr><td>Sensori-motor response</td><td>Acoustic startle</td><td>9a,b</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>↑ in -/-</td></tr><tr><td>Fear conditioning</td><td>%Time freezing</td><td>9e,f</td><td></td><td></td><td></td><td></td><td></td><td></td><td>↓ in -/-Y</td><td>↓ in +/-<br/>and -/-</td></tr></tbody></table>

Note: ↓ and ↑ arrows refer to a decrease or increase in the measured variable for each tested domain; phenotypes concerning +/- females, that is, reflecting the common clinical condition, are highlighted in blue.

Abbreviations: PND, post-natal day; USVs, ultrasonic vocalizations.

status of mice during development and adulthood (Caruso et al., 2020; Lahvis et al., 2011). Hence, the analysis of call composition, obtained through the classification of calls based on their duration and frequency characteristics (see also Figure S1 here), represents an important additional tool to assess meaningful ASD-relevant communicative phenotypes.

When the call type composition was specifically evaluated (Table 1), some common abnormalities were observed in Cdk15 mutant mice of both sexes during development. The proportion of “flat” calls was increased (although this difference emerged at different PNDs in the two sexes) while the percentage of “down” calls was reduced in both male and female (+/- and -/-) mutant pups; furthermore, male mutants emitted more “short” calls, in line with the overall reduced mean duration of their USVs. Interestingly, the same alterations in short,

down and flat calls were detected in mutant males at adulthood, thus suggesting a strong continuity of the pup and adult USV repertoire of male Cdk15 mice. In females, the alterations in call composition observed in mutants during development were instead not confirmed at adulthood, when only an increase in the proportion of complex calls was observed and exclusively in -/- mice. It is worth noticing that the same or highly similar call categories affected here by Cdk15 mutation were also altered in other genetic mouse models of ASD, for example, the BTBR (Scattoni et al., 2011) and the Fmr1-KO mouse (Kat et al., 2022); in particular, complex calls were also increased in Fmr1 mutant females (Gauducheau et al., 2017). Overall, our results support the view that the relative occurrence of different ultrasonic call types may have an important communicative and emotional value (de Chaumont et al., 2021; Kikusui et al., 2021;
Sangiamo et al., 2020) and may be indicative of patho-
logical phenotypes.

Nonetheless, caution is warranted to univocally inter-
pret the USV alterations observed here in pup and adult
mutants as autistic-like phenotypes; this prudent
approach should be applied not only to the *Cdk15*, but to
any ASD mouse model, because of the complexity of the
communication abnormalities described in ASD patients
and our actual limitations in modeling them in mice.
Although a reduction in the number of ultrasonic calls
has been for instance often reported in mouse models of
ASD (Ey et al., 2011; Scattoni et al., 2008; Scattoni
et al., 2011; Schmeisser et al., 2012), an exaggerated
ultrasonic call rate may also mimic ASD-relevant pheno-
types, as already suggested (Premoli et al., 2023). USV
rate in rodent pups indeed reflects also their affective
state (Branchi et al., 2001; Zimmerberg et al., 1994), with
an increase corresponding to an adverse emotional status
(Caruso et al., 2020). Higher pup USV rate has been also
described in some studies on mouse models of ASD
[reviewed in (Caruso et al., 2020)], where it has been
interpreted as a sign of enhanced emotional distress.
Interestingly, infants with ASD or at risk tend to emit
more cries than neurotypical children, especially at higher
fundamental frequencies that have been linked to more
aversive conditions and negative parental responses
(Esposito et al., 2017; Esposito & Venuti, 2010). Other
studies have instead interpreted the enhanced rate of
adult USVs described in some mouse models of ASD
as similar to the excessive talking and repetitive
speech sometimes described in autistic patients
[e.g., (Gauducheau et al., 2017; Wheeler, Raspa et al.,
2014)]. Despite the impressive advances in USV analysis
characterizing recent studies in laboratory mice (reviewed
in (Premoli et al., 2023)), we still need to fully understand
the precise communicative meaning of quantitative and
qualitative alterations in mouse ultrasonic calls. This
may be achieved by the combination of advanced tools
for USV analysis [e.g., (Chabout et al., 2015; Chabout
et al., 2017; Giua et al., 2024; Rusu et al., 2023)] in multi-
ple and more complex social settings with playback
approaches (Agarwalla et al., 2023; Asaba et al., 2015;
Beck et al., 2023; Hood et al., 2023; Hood &
Hurley, 2023; Niemczura et al., 2020); these strategies are
under active development in several research laborato-
ries, and will allow to optimize our interpretation of USV
phenotypes in mouse models of ASD and NDDs.

Sex-dependent autistic-relevant phenotypes in
*Cdk15*-KO mice

As already mentioned, the communication phenotype
tended to diverge between sexes (Table 1). Sex differences
were subtle during development, when only altered USV
duration was additionally detected in male mutants, but
became more marked in adulthood, when several call

types were altered in -/Y males, while only the propor-
tion of complex calls was different in -/- females. Fur-
thermore, no USV alterations were displayed by adult
+/- females. Similarly, -/- females were the only exper-
imental group showing increased sensory responsiveness
in the acoustic startle test, that is, a phenotype that has
been described in some other mouse models of ASD
(Haddad et al., 2023; Michalon et al., 2012; Zhang
et al., 2014) and that could resemble the hyperacusis
often reported in autistic patients (Ornitz et al., 1993;
Sinclair et al., 2017). Since only subtle sex differences
emerged overall, and they strictly depended on the tested
behavioral domain, it cannot be concluded that the phe-
notypic effects of the *Cdk15* mutation are overall more
marked in males. Taken together, the behavioral effects
of *Cdk15* mutation seemed therefore less sex-dependent
than those induced by other X-linked mutations, for
example, those involving the *Fmr1* gene (Gauducheau
et al., 2017; Pietropaolo & Subashi, 2014).

Despite the lack of a communication phenotype,
*Cdk15* +/− females indeed displayed several other ASD-
relevant behavioral alterations in adulthood, as marked
as those observed in −/Y males (Table 1); these included
enhanced stereotypic clasping behavior, reduced social
interaction, and memory deficits in the context freezing
test. The clasping phenotype is in line with previous find-
ings (Amendola et al., 2014; Gennaccaro et al., 2021;
Mottolese et al., 2023; Tassinari et al., 2022), although
our results allow to extend it to −/− females. Also the
social phenotype is in agreement with previous studies in
*Cdk15* mutant males (Jhang et al., 2017; Okuda
et al., 2018; Wang et al., 2012), and adds to the validity
of the *Cdk15* mouse line to model ASD-relevant behav-
iors (Crawley, 2007; Silverman et al., 2010). The social
effects of *Cdk15* mutation emerged in both sexes despite
the different social contexts used here for testing male
and female mice: indeed, same-sex social encounters were
performed in females, while they could not be assessed in
males because of the well-known limitation of intermale-
aggression. Nonetheless, sociability could be similarly
evaluated in both sexes, despite the different social testing
conditions, as sexual behaviors were prevented in male
mice by the specific experimental settings we used here,
as in previous studies in other mouse models of NDDs
(e.g., (Fyke et al., 2021; Premoli et al., 2022)). These set-
tings include the short duration of the session, its occur-
rence during the light phase of the day and the use of
stimulus females in non-receptive estrous phase, resulting
in the absence of mounting attempts by male subjects.

Similarly, the deficits in context freezing, are in line
with previous findings on this (Fuchs et al., 2022;
Gennaccaro et al., 2021; Wang et al., 2012) and other
ASD models [e.g., (Baker et al., 2010; Gaudissard
et al., 2017)]. The hyperactivity displayed by mutant mice
of both sexes could represent a potential confounding
factor affecting their freezing levels. Nonetheless, our
findings from Day 1 showed no difference between
mutant and WT males or females in their freezing levels during the post-shock interval (Figure 9c,d), thus supporting the presence of a genuine deficit in context memory formation; this interpretation was further supported by the analysis of the time-pattern of freezing response on Day 2, since this revealed (especially in male mice) a time-bin specific reduction in freezing time in *Cdkl5* mutants (Figure 9g,h). Furthermore, the context freezing deficits observed on testing Day 2 were detected in the absence of genotype differences in learning abilities during the conditioning phase (Day 1), thus further supporting the occurrence of reduced hippocampal-dependent memory in mutant mice. Indeed, several studies have described cognitive deficits in *Cdkl5* male and female mutants in different hippocampal-dependent tasks (Fuchs et al., 2015; Fuchs et al., 2022; Fuchs, Fustini, et al., 2018; Fuchs, Gennaccaro, et al., 2018; Gennaccaro et al., 2021; Tang et al., 2017; Wang et al., 2012).

It is important to underline that cortical visual impairment, a phenotype frequently described in CDD patients (Quintiliani et al., 2021) probably linked to a defective maturation of excitatory and inhibitory visual circuits (Amendola et al., 2014; Mazziotti et al., 2017), has been reported in both male and female *Cdkl5*-KO mice (Lupori et al., 2019; Mazziotti et al., 2017). In mutants, deficits in the visual response amplitude, reduced visual acuity and defective contrast function were observed starting at PND 27-28, and they persist into adulthood (i.e., at 2-3 months of age) (Mazziotti et al., 2017). Hence, a contribution of visual deficits can be ruled out for the behavioral alterations displayed here by *Cdkl5* mutant pups before weaning (i.e., USVs and exploration), while it could be a confounding factor for the behaviors assessed in adult mutants. Nonetheless, the behavioral impact of cortical visual impairment in adult male and female *Cdkl5* mutants seems limited in cognitive tests, as it was not accompanied by altered ability to find a visible platform in the water maze (Hao et al., 2021). Nonetheless, studies with *Cdkl5* conditional mutant and/or genetic rescuing approaches (Gao et al., 2020; Lupori et al., 2019; Silvestre et al., 2024; Tang et al., 2017; Terzic et al., 2021) should be performed in the future to disentangle the contribution of visual deficits to the explorative, social, acoustic and cognitive phenotypes of *Cdkl5* mice.

## Conclusions

Our findings support the validity of the *Cdkl5* mouse line to model ASD-relevant phenotypes during development and in adulthood, thus encouraging its use in preclinical studies to validate the potential of therapeutic strategies for ASD and CDD. Furthermore, our results provide additional evidence supporting the ability of *Cdkl5* +/− females, representing the common clinical condition of CDD patients, to model a relevant pathological phenotype

of this disorder (Fuchs et al., 2022; Fuchs, Gennaccaro, et al., 2018). The inclusion of female subjects in preclinical neurobehavioral studies is an issue of high general relevance, which is receiving particular attention in the context of ASD. Despite a traditional focus on the male sex, ASD research is indeed increasingly interested in evaluating pathological behavioral phenotypes also in female subjects, as ASD female patients may have unique clinical presentations relative to their male counterparts, a factor that may have led to under diagnosis of ASD in the female sex (Loomes et al., 2017).

## AUTHOR CONTRIBUTIONS

Experimental activity, data collection, and analysis: N.M., O.C., C.F., A.M., and S.P. Study design and project supervision: E.C. and S.P. Manuscript writing: N.M., A.M., E.C., and S.P. All authors have reviewed and approved the manuscript.

## ACKNOWLEDGMENTS

The authors thank the genotyping facility of Neurocentre Magendie, funded by Inserm and LabEX BRAIN ANR-10-LABEX-43, for animal genotyping. We thank Elodie Poinama and Valeria Petroni for animal care, Marie-Laure Rousseau and Thierry Lafon for administrative and technical support.

## CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

## DATA AVAILABILITY STATEMENT

Data are available upon reasonable request.

## ETHICS STATEMENT

All experimental procedures were in accordance with the European Communities Council Directive 2010/63/EEC, and approved by local ethical committee (“Comité d’Ethique pour l’experimentation animale de Bordeaux”, CE 50) and the French Ministry (“Ministère de l’enseignement supérieur de la recherche et de l’innovation”).

## ORCID

Susanna Pietropaolo https://orcid.org/0000-0002-8993-1922

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## SUPPORTING INFORMATION

Additional supporting information can be found online in the Supporting Information section at the end of this article.

**How to cite this article:** Mottolese, N., Coiffard, O., Ferraguto, C., Manolis, A., Ciani, E., & Pietropaolo, S. (2024). Autistic-relevant behavioral phenotypes of a mouse model of cyclin-dependent kinase-like 5 deficiency disorder. *Autism Research*, 1–18. https://doi.org/10.1002/aur.3226