Second occurrence of the PAK3-R67C variation and multiscale analysis of the corresponding knock-in mice reveal novel phenotypic features and functional synaptic defects

Manon Dobrigna, Sandrine Poëa-Guyon, Mathieu Legras, Delphine Le Verger, Kévin Duarte, Catherine Sébrié, Roseline Poirier, Sylvie Granon, Serge Marty, Annick Toutain, et al.

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To cite this version:

Manon Dobrigna, Sandrine Poëa-Guyon, Mathieu Legras, Delphine Le Verger, Kévin Duarte, et al. Second occurrence of the PAK3-R67C variation and multiscale analysis of the corresponding knock-in mice reveal novel phenotypic features and functional synaptic defects. Neurobiology of Disease, 2025, 217, pp.107188. 10.1016/j.nbd.2025.107188. hal-05370772

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# Second occurrence of the PAK3-R67C variation and multiscale analysis of the corresponding knock-in mice reveal novel phenotypic features and functional synaptic defects

Manon Dobrigna<sup>a</sup>, Sandrine Poëa-Guyon<sup>a</sup>, Mathieu Legras<sup>a</sup>, Delphine Le Verger<sup>a</sup>, Kevin Duarte<sup>a</sup>, Catherine Sébrié<sup>b</sup>, Roseline Poirier<sup>a</sup>, Sylvie Granon<sup>a</sup>, Serge Marty<sup>c</sup>, Annick Toutain<sup>d</sup>, Glenn Dallérac<sup>a</sup>, Cyrille Vaillend<sup>a</sup>, Jean-Vianney Barnier<sup>a,∗</sup>

## ARTICLE INFO

**Keywords:**

Intellectual disability

Mouse knock-in

Genotype/phenotype correlation

Microcephaly

PAK3-R67C

## ABSTRACT

Variations in PAK3, a gene located on the X chromosome, are known to contribute to intellectual disability (ID) and are associated with a broad clinical spectrum. The correlation between genotype and phenotype in PAK3-related disorders remains incompletely understood. In this study, we focused on the PAK3-R67C variant, the most amino-terminal variation identified to date, which was initially associated with a non-syndromic form of X-linked ID. We describe the clinical picture of its second occurrence, and present additional clinical insights. Our findings indicate that the R67C mutation is associated with moderate to severe ID, along with speech and gait abnormalities, and features consistent with autism spectrum disorders. To explore these associations further, we conducted a comprehensive phenotypic analysis using a corresponding knock-in mouse model. The Pak3-R67C mice, which present significant microcephaly, displayed mild motor deficits, social avoidance behaviour, and impaired emotional learning and memory. At the physiological level, Pak3-R67C mice exhibited reduced hippocampal synaptic efficacy and defects in long-term potentiation, despite unaltered synapse structure. In vitro biochemical assays revealed that the PAK3-R67C variant enhances interactions with PAK-interacting exchange factors (PIX). Notably, the expression of this variant in COS7 cells, altered the distribution of focal adhesions, while cell migration capacity remained unaffected. Collectively, these data, which include the second reported occurrence of the R67C variant, a detailed characterization of the corresponding knock-in mouse line, and a multi-tiered analysis of its biological and biochemical properties, contribute to a better understanding of the genotype-phenotype relationship in PAK3-related disorders.

## 1. Introduction

The group I p21-activated kinases PAK1, PAK2, and PAK3 are serine/threonine kinases with a wide range of substrates involved in the regulation of various signalling pathways, particularly in the control of actin cytoskeleton dynamics (Bokoch, 2003). In brain, these kinases play key roles in neural precursor proliferation, cell fate determination, neuronal differentiation, migration, axonal specification, and dendritic arborization (Kreis et al., 2007). They are also crucial for dendritic spine formation and dynamics, as well as overall synaptic plasticity (Boda

et al., 2004; Hayashi et al., 2004). Although these three kinases share a high degree of sequence identity, numerous substrates and partners, and fundamental regulatory mechanisms, they exhibit distinct cellular and developmental expression patterns, allowing them to fulfil specific functions in the brain. Genetic variations affecting the group I PAK genes have been linked to neurodevelopmental disorders (NDDs), each associated with distinct clinical manifestations (Dobrigna et al., 2023). Pathogenic PAK1 mutations are gain-of-function variants associated with intellectual developmental disorder with macrocephaly, seizures, and speech delay (IDDMSSD, OMIM #6181586) (Harms et al., 2018;

<footnote><sup>a</sup> Université Paris-Saclay, CNRS UMR9197, Institut des Neurosciences Paris-Saclay, 91400 Saclay, France</footnote>

<footnote><sup>b</sup> Université Paris-Saclay CNRS, CEA, Laboratoire d'Imagerie Biomédicale Multimodale (BioMaps), Service Hospitalier Frédéric Joliot, 91401 Orsay, France</footnote>

<footnote><sup>c</sup> Sorbonne Université, Institut du Cerveau - Paris Brain Institute - ICM, Inserm, CNRS, APHP, Hôpital de la Pitié Salpetrière, Paris, France</footnote>

<footnote><sup>d</sup> University Hospital of Tours, Department of Genetics, UMR1253, iBrain, INSERM, Tours, France</footnote>

<footnote>* Corresponding author.
E-mail address: jean-vianney.barnier@cnrs.fr (J.-V. Barnier).</footnote>
Scorrano et al., 2023). In contrast, PAK2 variations are loss-of-function mutations leading to PAK2 haplo-insufficiency and are associated with intellectual disability (ID), autism spectrum disorders (ASD), retinal degeneration, and encephalocele, corresponding to Knobloch syndrome type II (OMIM #618458) (Antonarakis et al., 2021; Werren et al., 2024).

The case of PAK3 is more complex as approximately twenty different PAK3 variants have been described, linked to variable degrees of X-linked intellectual disability (XLID) and additional clinical manifestations, including autistic traits, attention deficit hyperactivity disorder (ADHD), epilepsy, seizures, and brain abnormalities such as microcephaly or macrocephaly (Dobrigna et al., 2023; Pascolini et al., 2021; Gambardella et al., 2025). Currently, there is no simple genotype-phenotype correlation for PAK3-related disorders. One hypothesis is that different PAK3 variations affect distinct protein functions, leading to diverse pathogenic mechanisms associated with specific clinical features. For example, some missense variants, such as K389N or G424R, result in severe to lethal phenotypes, as they lead to kinase-deficient PAK3 proteins, disrupting ERK- or PIX-dependent signalling pathways (Duarte et al., 2020; Iida et al., 2019; Magini et al., 2014; Nagy et al., 2020; Pascolini et al., 2021; Qian et al., 2020). Furthermore, nonsense mutations that are responsible for mild to moderate ID result in nonsense-mediated mRNA decay (NMD) of PAK3 transcripts, as recently demonstrated (Nicolas-Martinez et al., 2024). Decreases in protein expression due to NMD, or to instability of the mutated protein as in the case of the A365E variant, lead to a loss of PAK3 function and are responsible for the less severe PAK3-related diseases (Allen et al., 1998; Gedeon et al., 2003).

In contrast, the R67C variant, the first PAK3 missense mutation identified in the MRX47 family, leads to moderate to severe ID (Bienvenu et al., 2000; des Portes et al., 1997). The R67C variation is the most amino-terminally located mutation described to date. Positioned outside the catalytic domain, this mutation does not alter the intrinsic kinase activity of PAK3 (Kreis et al., 2007). More specifically, the PAK3-R67C mutation is located in a lysine-enriched region, just upstream of the CDC42/RAC-interacting domain (CRIB) within the regulatory region of PAK3. This polybasic region is critical for interactions with GTPases, playing a key role in defining GTPase specificity, affinity, and activation (Knaus et al., 1998). Importantly, the PAK3-R67C mutation significantly alters the binding properties of PAK3: it enhances its interaction with RAC1 while reducing its affinity for CDC42, likely leading to changes in kinase activation dynamics (Kreis et al., 2007). Additionally, this variant exhibits decreased affinity for NCK2, an adaptor protein rich in SH3 domains, whose interaction with PAK3 is crucial for regulating synaptic transmission (Thévenot et al., 2011). However, PAK3-R67C does not affect PAK3 heterodimerization with PAK1, as it is located outside the region involved in PAK dimerization (Combeau et al., 2012). To further investigate the phenotype associated with this variation, we previously developed the first mouse model carrying the Pak3-R67C variant (Castillon et al., 2020). Our original cognitive assessments revealed that adult male Pak3-R67C mice exhibited selective impairments in long-term spatial memory and pattern separation, which were attributed to altered adult hippocampal neurogenesis (Castillon et al., 2020).

In this study, we further elucidate the genotype-phenotype correlation associated with the R67C variant. We report here the clinical data of the patients with the second occurrence of this mutation in patients with ID, enabling us to expand the first clinical description. Additionally, we conducted an extensive phenotypic screening of the corresponding knock-in mouse model, which led to the identification of new phenotypic traits, including mild motor strength deficits, cognitive and social impairments, and microcephaly. We also identified structural and functional synaptic defects in the hippocampus, along with biochemical and cellular alterations that may explain the observed mouse phenotype. Collectively, these clinical and experimental findings confirm the causal role of the PAK3-R67C variation in the associated symptoms and validate the robustness of the animal model for studying PAK3-related disorders.

## 2. Results

### 2.1. New clinical features associated with the PAK3-R67C pathogenic variation

Two occurrences of the R67C variant have been reported in the literature (Fig. 1A,B) (Bienvenu et al., 2000; Grozeva et al., 2015). The first description involved six males across two generations within a single family MRX47. All presented moderate to severe ID without seizures or behavioural problems. Three affected individuals were clinically assessed and had no other major clinical features, particularly no growth problems or physical anomalies such as dysmorphic features or congenital malformations. Two patients (III-2 and IV-7) exhibited very limited verbal communication. The phenotype was classified as non-syndromic ID (des Portes et al., 1997) (Table 1). As the family was then lost for follow-up, no additional information concerning the clinical signs could be obtained since this publication. The second occurrence was identified in a large cohort study involving approximately 1000 patients, which aimed to uncover genetic causes of ID by screening a panel of around 500 known or candidate ID-associated genes using targeted next-generation sequencing (Grozeva et al., 2015). Here, we report for the first time, the clinical features of three affected individuals from family #575. Late milestones in motor skills were achieved in the proband. He had very slow speech and his language remained very poor. He had learning difficulties with low ability in reading and writing, and attended a special school. He also had behavioural problems with a poor sense of danger, and difficulties in social interactions with poor eye contact and difficulties in engaging with others. His brother also had a delay in motor skills and delayed speech. He also attended a special education school for learning disability. He had obsessive traits and marked repetitive behaviour. The same clinical picture than in the proband was described in a maternal uncle. No morphological features such as congenital malformations, dysmorphic traits or microcephaly were described in these individuals. In summary, in this family, key characteristics include global delay with a speech delay which was noticeably more marked than the motor delay, moderate to severe ID, and autistic traits (Table 1). Taken together, these two clinical reports suggest that the phenotypic spectrum associated with the R67C variant differs from those observed in cases involving nonsense variants or variants that abolish kinase activity (Allen et al., 1998; Magini et al., 2014; Duarte et al., 2020).

### 2.2. Mild motor strength defects in Pak3-R67C mice

Gait abnormalities and oro-motor dysfunctions, such as drooling and articulation or speech impairments, have been frequently reported in patients with PAK3 mutations, including in the second reported case of the R67C variant (Dobrigna et al., 2023; Duarte et al., 2020; Pascolini et al., 2021). Based on these clinical observations, we assessed motor impairments in the corresponding Pak3-R67C knock-in mouse model using the traction reflex test, wire grid, and Rotarod tests. In the traction reflex test, mice were individually placed with their forepaws on a horizontal wire, and the latency to exhibit a traction reflex, to fall, or to escape laterally was recorded. Performance was scored according to predefined criteria (see Materials and Methods). No differences were observed in the latency to elicit a traction reflex between genotypes (Fig. 2A). However, Pak3-R67C mice exhibited a significantly shorter latency to fall (Fig. 2B). Furthermore, a significantly greater proportion of R67C mice fell during the test (86.67 %) compared to wild-type (WT) controls (16.67 %) across the three sessions. Accordingly, the overall performance scores were lower in Pak3-R67C mice than in WT littermates (Fig. 2C). Additionally, escape latencies were longer in Pak3-R67C than in WT mice (Fig. 2D). Grip strength was assessed using the wire grid test, in which mice were suspended upside-down by all four limbs and the in latency to fall was measured. Unlike in the traction test, escape was not possible. In this test as well, Pak3-R67C mice exhibited
<figure><img src="image_4.png" /><figcaption>Fig. 1. Genetic trees and clinical traits of the two PAK3-R67C occurrences.</figcaption></figure>

(A) Family MRX47. Data extracted from des Portes et al., 1997. Dark filled colour represents the affected males.

(B) Family 575, cited in the article from Grozeva et al., 2015. The arrow points the proband.

<caption>**Table 1**</caption>

Clinical data of patients from the two family.

<table><thead><tr><th colspan="7">Dobrigna et al., NBD 25-889R3</th></tr><tr><th colspan="7">PAK3-R67C variation</th></tr><tr><th></th><th colspan="3">Family MRX47</th><th colspan="3">Family #575</th></tr></thead><tbody><tr><td>Clinical description</td><td colspan="3">des Portes et al., 1997</td><td colspan="3">L. Raymond, unpublished data</td></tr><tr><td>Genetic description</td><td colspan="3">Bienvenu et al., 2000</td><td colspan="3">Grozeva et al., 2015</td></tr><tr><td>ClinVar RCV</td><td colspan="3">RCV000012325</td><td colspan="3">RCV005621848</td></tr><tr><td>ID (degree)</td><td>III-2<br>Moderate/severe</td><td>III-4</td><td>IV-7</td><td>I-2</td><td>II-1</td><td>II-2</td></tr><tr><td>Delayed motor skills</td><td>Not reported</td><td>Moderate</td><td>Moderate</td><td>Moderate/severe</td><td>Moderate/severe</td><td>Moderate/severe</td></tr><tr><td>Speech/language defect</td><td>Poor language</td><td>Language impairment</td><td>Poor language</td><td>Delayed walking</td><td>Walking at 18 months</td><td>Walking at 17 months sitting at 11 months</td></tr><tr><td>Reading, writing</td><td>No</td><td>A few words</td><td>No</td><td>Slow speech; poor language</td><td>Slow speech; poor language</td><td>Delayed speech</td></tr><tr><td>Social Interaction</td><td></td><td></td><td></td><td>Poor</td><td>Poor</td><td></td></tr><tr><td>Stereotypies</td><td></td><td></td><td></td><td>Poor</td><td>Poor</td><td></td></tr><tr><td>Obsessive traits</td><td></td><td></td><td></td><td>No</td><td>No</td><td></td></tr><tr><td>Microcephaly</td><td>Not reported</td><td>Not reported</td><td>Not reported</td><td>No</td><td>No</td><td></td></tr></tbody></table>

significantly shorter fall latencies than WT controls (Fig. 2E). In contrast, performance on the Rotarod test did not reveal any notable deficits. Pak3-R67C mice performed similarly to WT controls at constant speeds (0 rpm and 4–16 rpm), which require coordination and balance (Fig. 2F), as well as in the accelerated version of the Rotarod test (4–40 rpm over 180 s) (Fig. 2G). Performance improved similarly across three consecutive days in both groups. Overall, these results indicate that Pak3-R67C mice exhibit mild but statistically significant grip strength impairments, while coordination and balance remain unaffected, suggesting that the R67C variation affects muscle strength rather than cerebellum-dependent motor functions.

## 2.3. Pak3-R67C mice present social and cognitive defects

Given recent clinical data indicating behavioural abnormalities in patients carrying the R67C variant, we investigated the behavioural

phenotype of the Pak3-R67C knock-in mouse model. To assess cognitive function, we conducted a battery of tests including auditory cued and contextual fear conditioning, and spatial object recognition. In the auditory cued fear-conditioning task, Pak3-R67C mice performed similarly to wild type (WT) controls (Fig. 3A). No significant differences were observed between genotypes in the memory retention test conducted 24 h later (Fig. 3B). For contextual fear conditioning, mice were placed in a conditioning chamber for 8 min, receiving three foot shocks spaced by two-minute intervals following a two-minute acclimation period. Pak3-R67C mice exhibited a learning delay, with freezing behaviour reaching only 37 % of test duration compared to 55 % in WT mice by the end of the acquisition session (Fig. 3C). We then tested retention of fear memory by placing the mice in the same context during successive retention sessions spaced by 24 h intervals. Pak3-R67C mice showed reduced freezing behaviour during these sessions, thus indicating impaired memory retention (Fig. 3D). These results suggest that Pak3-
<figure><img src="image_5.png" /><figcaption>Fig. 2. Assessment of Pak3-R67C motor functions.</figcaption></figure>

(A–D) Traction test.

(A). Latency until first traction (n = 12 WT, 15 R67C; Wilcoxon Mann-Whitney, NS).

(B) Latency until fall (Wilcoxon Mann-Whitney, *p* = 0.0005).

(C) Traction score - cf. material & methods (Wilcoxon Mann-Whitney, *p* = 0.0028).

(D) Latency until escape (Student t-test, *p* = 0.0231).

(E) Inverted grid: latency until fall (n = 12 WT, 15 R67C, Wilcoxon Mann-Whitney, *p* = 0.0439)

(F) Latency until fall, at different speed of the rotarod (static – 0 rotation/min, 4 rotations/min and 16 rotations/min). Clear lines represent each individual, bold lines represent the mean for each genotype (n = 15 WT, 12 R67C, 2-way ANOVA, time factor: F (1.793, 43.03) = 219.4; *p* < 0.0001; genotype factor: F (1, 24) = 0.6628, NS; time x genotype interaction: F (2, 48) = 0.7244, NS).

(G) Latency until fall, in the accelerating rotarod, across 3 consecutive days. (4 to 40 rotations/min), Clear lines represent each individual, bold lines represent the mean for each genotype. 2-way ANOVA; time factor: F (1.609, 40.21) = 10.09, *p* = 0.0007; genotype factor: F (1, 25) = 0.04319, NS; time x genotype interaction: F (2, 50) = 0.8928, NS.

R67C mice have impaired acquisition of contextual fear memory, leading to reduced recall performance. Spatial learning and memory were evaluated using the spatial object recognition test. Both genotypes explored objects equally during the acquisition phase (Fig. 3E). In the retention session 24 h later, both groups showed a clear preference for the displaced object, with discrimination ratios significantly above chance (33 %) and no differences between genotypes (Fig. 3F), indicating intact spatial recognition memory.

Social behaviour was assessed using the social interaction test (SIT), during which a novel adult male mouse was presented to the test mouse in a neutral environment (de Chaumont et al., 2012; Martucci et al., 2023). No significant differences were observed between Pak3-R67C and WT mice in the duration of social contacts (Fig. 3G), the duration of affiliative contact (Fig. 3H) or in the number of dominant behaviours (e. g., paw control) (Fig. 3I). However, Pak3-R67C mice presented significantly more avoidance behaviour, fleeing from social contact more frequently than WT littermates (Fig. 3J). However, this was not associated with a difference in the frequency at which test mice were pursued by the visitor mouse (Fig. 3K).

To evaluate anxiety-like behaviours, we used the elevated plus maze and found no significant differences between genotypes in exploratory activity (Supplementary Fig. 1A). Resignation-like behaviours were

assessed using the tail suspension test. Both genotypes displayed similar immobility latencies (approximately 33 s in the first session and 20 s in the second session, conducted 24 h later), with no differences in immobility duration across sessions (Supplementary Fig. 1B,C). The forced swim test yielded comparable results, with no differences in immobility latency or duration between genotypes (Supplementary Fig. 1D,E). We further measured the startle response to increasing sound pulses and the inhibition of this response by a weak, non-startling prepulse. No differences were found between genotypes in the amplitude and latency of startle responses, nor in the prepulse inhibition (Supplementary Fig. 1F,G).

In summary, Pak3-R67C mice exhibit mild cognitive impairments during associative learning tasks, particularly in contextual fear conditioning, while spatial memory remains unaffected. Social behaviours were largely preserved, although Pak3-R67C mice displayed increased avoidance in response to unfamiliar adult males, suggesting altered social reactivity rather than general social withdrawal or aggression. Importantly, these cognitive and social abnormalities occurred in the absence of differences in anxiety-like behaviour, stress responses, or sensorimotor gating. These findings indicate that the R67C variant selectively impacts associative learning and specific aspects of social interaction.
<figure><img src="image_6.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>E</figcaption></figure>

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

<figure><img src="image_9.png" /><figcaption>I</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>B</figcaption></figure>

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

<figure><img src="image_12.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>J</figcaption></figure>

<figure><img src="image_15.png" /><figcaption>K</figcaption></figure>

<figure><img src="image_16.png" /></figure>

(caption on next page)
<caption>**Fig. 3.** Behaviour and cognition anomalies of *Pak3-R67C* mice.</caption>

(A) Percentage of freezing time recorded during auditory-cued fear conditioning acquisition (n = 14 WT, 12 R67C, 2-way ANOVA; CS factor: F (2.824, 67.78) = 49.31, *p* < 0.0001; genotype factor: F (1, 24) = 2.993, NS; Cs x genotype interaction: F (4, 96) = 0.8009, NS.
(B) Percentage of freezing time recorded during auditory-cued fear conditioning retention (n = 14 WT, 12 R67C, 2-way ANOVA: CS factor: F (2.454, 58.89) = 5.749, *p* = 0.0030; genotype factor: F (1, 24) = 0.04007, NS; Cs x genotype interaction: F (3, 72) = 0.7552, NS.
(C) Percentage of freezing time recorded during contextual fear conditioning acquisition, (n = 12 WT, 15 R67C, 2-way ANOVA; time factor: F (2.548, 56.06) = 63.99, *p* < 0.0001; genotype factor: F (1,22) = 4.802, *p* = 0.0393; time x genotype interaction: F (3, 66) = 3.662, *p* = 0.0166; multiple comparisons Benjamini, Krieger and Yekutieli, 8 min q = 0.0099).
(D) Percentage of freezing time during the first 2 min of each retention session (without US). 2 way ANOVA; retention session factor: F (2.380, 52.35) = 14.40, *p* < 0.0001; genotype factor: F (1, 22) = 7.304, *p* = 0.0130; retention session x genotype interaction: F (3, 66) = 0.1991, NS.
E-F: Object location task (n = 13 WT, 11 R67C).
(G) Time of contact between mice during SIT (Student *t*-test, NS).
(H) Time of affiliative contact between mice (Student *t*-test, NS).
(I) Number of paw control from the test mouse (Wilcoxon Mann-Whitney, NS).
(J) Number of times the test mouse escaped from a potential contact initiated by the visitor mouse (Wilcoxon Mann Whitney, *p* = 0.0261).
(K) Time spent by visitor mice pursuing test mice during SIT (Wilcoxon Mann-Whitney, NS).

## 2.4. Adult *Pak3-R67C* mice present functional synaptic defects

Since our previous characterization of cognitive functions revealed hippocampus-dependent deficits (Castillon et al., 2020), we investigated potential electrophysiological abnormalities within the dorsal hippocampus. Given the high expression of PAK3 in the CA1 region, we examined neurotransmission at Schaffer collateral synapses by recording field excitatory postsynaptic potentials (fEPSPs). To determine whether the *Pak3-R67C* mutation affects basal synaptic transmission, we performed input/output experiments by recording fEPSPs at increasing stimulation intensities. The resulting input/output curves indicate that *Pak3-R67C* mice exhibit significantly reduced basal synaptic transmission (Fig. 4A). To test whether this reduction could be attributed to altered excitability of the Schaffer collaterals, we measured the input/output relationship of the afferent volley amplitude and observed no difference between *Pak3-R67C* and WT mice (Fig. 4B). Thus, the decreased fEPSP slope appears to result from reduced synaptic efficacy. To further assess the origin of this defect, we performed paired-pulse experiments, delivering two successive stimuli separated by short incremental intervals, to evaluate presynaptic function, as paired-pulse facilitation is an established index of neurotransmitter release probability (Zucker, 1973; Zucker and Regehr, 2002). No differences were found in the paired-pulse ratio between *Pak3-R67C* and WT mice (Fig. 4C), suggesting that the probability of neurotransmitter release is unaltered. Together, these experiments indicate that the *Pak3-R67C* mutation induces a postsynaptic impairment of hippocampal neurotransmission. Because long-term synaptic plasticity is considered a fundamental substrate for learning and memory, we then assessed long-term potentiation (LTP) at the Schaffer collateral CA3-to-CA1 synapses. Strikingly, *Pak3-R67C* mice displayed a marked impairment in LTP induction, as well as a notably reduced duration of LTP compared to WT mice (Fig. 4D). In summary, these results demonstrate that the *Pak3-R67C* mutation leads to significant deficits in both basal synaptic transmission and long-term synaptic plasticity in the CA1 region of the hippocampus, likely owing to postsynaptic dysfunction, which may underlie some of the cognitive deficits previously reported in this model.

## 2.5. Structural synaptic anomalies in the hippocampus of *Pak3-R67C* mice

Considering that the *Pak3-R67C* variant leads to functional alterations in the hippocampus, and since previous studies showed that postnatal PAK inhibition leads to structural synaptic defects whereas *Pak3*-knock-out does not, we aimed to further ascertain synaptic

alterations by performing ultrastructural analyses using electron microscopy (Boda et al., 2004; Hayashi et al., 2004; Meng et al., 2005). First, we measured the width of the *Stratum Radiatum* in semi-thin sections, which corresponds to the CA1 region where Schaffer collaterals connect to pyramidal neurons (Fig. 5A), and observed no difference between *Pak3-R67C* mice and WT littermates (Fig. 5B). Neuron density was then quantified in the *Stratum Pyramidale* (Fig. 5C,D) and was found to be significantly higher by 10.6 % in *Pak3-R67C* mice (Fig. 5E). However, we found no change in synapse density in ultrathin sections of the *Stratum Radiatum* between WT and *Pak3-R67C* mice (Fig. 5F-H). Finally, the size of post-synaptic densities (PSD) were not affected either (Fig. 5I), suggesting that the electrophysiological alterations are most likely not associated with post-synaptic structural changes. Furthermore, the increase in neuron density, but not in synapse density could indicate a reduction of synapse number per neuron in the CA1 region of the hippocampus of *Pak3-R67C* mice, which might well underlie the decreased neurotransmission assessed at the field level.

## 2.6. *Pak3-R67C* mice exhibit microcephaly

Throughout the aforementioned experiments, we consistently observed that the brains of male *Pak3-R67C* mice were smaller than those of their WT littermates (Fig. 6A). Notably, although there was no significant difference in overall body weight, the brain weight of *Pak3-R67C* mice was significantly reduced compared to WT controls (Supplementary Fig. 2A,B). A 13.7 % decrease in the brain-to-body weight ratio was also observed in *Pak3-R67C* mice relative to their WT counterparts (Fig. 6B). Magnetic resonance imaging (MRI) of anaesthetised male mice aged 4 to 6 months revealed a 13.1 % reduction in the total brain volume- in *Pak3-R67C* animals (Fig. 6C). Furthermore, 3D MRI reconstructions of WT and *Pak3-R67C* brains enabled volumetric analysis of individual brain regions (Fig. 6D). The different regions of the *Pak3-R67C* brains displayed a volume reduction, of approximately 13 % in cortex, 13.8 % in striatum, 31 % in ventricles, 19.5 % in hippocampus, and 9.9 % in cerebellum (Fig. 6E). Collectively, these findings indicate that *Pak3-R67C* mice exhibit microcephaly, characterized by a 10 to 19.5 % volume reduction across multiple brain regions, with the most pronounced decrease observed in the hippocampus.

## 2.7. *PAK3-R67C* disruption of cellular biochemical processes

Previous in vitro studies have shown that several biochemical defects may be responsible for phenotypes, such as change in kinase activity, protein stability, or partner affinity (Allen et al., 1998; Duarte et al.,
<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /><figcaption>**Fig. 4.** Functional synaptic defects of Pak3-R67C mice.</figcaption></figure>

(A) fEPSP slopes expressed as a percentage of the mean recorded in the WT group at 70 V (n = 11 WT and 26 slices, 9 R67C and 16 slices. 2-way ANOVA: stimulus intensity factor: F (1.431, 61.51) = 90.27, *p* < 0.0001; genotype factor: F (1, 43) = 2.883, NS; stimulus intensity x genotype interaction: F (10, 430) = 1.966, *p* = 0.0355. Above the graph are example traces of fEPSP generated with a 70 mV, 40 µs stimulus.

(B) Afferent volley amplitude of both genotypes expressed as a percentage of WT mean at 70 V (n = 11 WT and 26 slices, 9 R67C and 16 slices. 2-way ANOVA, stimulus intensity factor: F (1.110, 47.73) = 96.40, *p* < 0.0001; genotype factor: F (1, 43) = 0.01129, NS; stimulus intensity x genotype interaction: F (10, 430) = 0.3111, NS.

(C) Short-term potentiation of the second fEPSP relative to the first at various time intervals between paired stimulations. Above the graph are example traces of paired pulses at 50 ms interval (9 WT and 17 slices, 10 R67C and 21 slices. 2-way ANOVA; stimulus interval factor: F (1.467, 52.82) = 66.97, *p* < 0.0001; genotype factor: F (1, 36) = 0.1509, NS, genotype x stimulus intensity interaction: F (3, 108) = 0.3793, NS).

(D) fEPSP percentage of change compared to baseline before and after induction of long-term potentiation (3 HFS of 100 Hz). Above the graph are example traces of 30s before tetanus (1), 30s after tetanus (2) and 110 min after tetanus (3), (6 WT and 6 slices, 5 R67C and 7 slices, 2-way ANOVA; time factor: F (3.846, 42.30) = 22.08, *p* < 0.0001; genotype factor: F (1,11) = 1.574, NS; time x genotype interaction: F (110,1210) = 1.654, *p* < 0.0001.

2020; Kreis et al., 2007; Magini et al., 2014). Specifically, the PAK3-R67C variant exhibits altered affinities for its primary activators, RAC1 and CDC42, resulting in partial impairment of its kinase activation (Kreis et al., 2007). We sought to identify additional biochemical defects

associated with the PAK3-R67C mutation. First, we evaluated the PAK3-R67C variant stability, as it was shown that some Pak3 variations, such as Pak3-A365E, destabilize the protein and did not observe significant instability of the PAK3-R67C protein expressed in COS7 cells (data not
<figure><img src="image_21.png" /><figcaption>**Fig. 5.** Structural and ultrastructural anomalies of hippocampal CA1 neurons of *Pak3-R67C* mice.</figcaption></figure>

(A) Semithin section in the CA1 *stratum radiatum* of WT mice. The double arrows indicate measurements of the height of the *stratum radiatum*. The scale bar is 100 µm.

(B) Height of *stratum radiatum* in WT and R67C mice (n = 6 WT, 4 R67C, 3 slices per individual, unpaired t-test, NS).

(C-D) Semithin section in the CA1 *stratum pyramidale* of WT and R67C mice respectively. White arrows point to neuronal nuclei, the red arrow indicates a microglial nucleus, and the yellow arrow points to an astrocytic nucleus. The scale bar is 50 µm.

(E) Neuron number in a 310 µm width band (n = 6 WT, 4 R67C, 3 slices per individual, unpaired t-test, *p* = 0.0009).

(F) Ultrathin section in the CA1 *stratum radiatum* of WT mice. Arrows point to postsynaptic densities. The scale bar is 500 nm.

(G) Ultrathin section in the CA1 *stratum radiatum* of R67C mice. Arrows point to postsynaptic densities. The scale bar is 500 nm.

(H) Number of synapses in a 53 µm² field of *stratum radiatum* (n = 8 WT, 7 R67C, unpaired t-test, NS).

(I) Frequency distribution of PSD lengths in WT and R67C mice *stratum radiatum* (n = 6 WT, 4 R67C, 100 PSDs per individual; 2-way ANOVA; PSD length factor: F (14, 112) = 62.30, *p* < 0.0001, genotype factor: F (1, 8) = 0.08421, NS; PSD length x genotype interaction: F (14, 112) = 0.9697, NS).

shown) (Duarte et al., 2020; Magini et al., 2014).

PAK3/PIX binding has been shown to increase for variants associated with severe cases of ID and microcephaly (Duarte et al., 2020). To examine whether the R67C variation alters PAK3/PIX binding, we first performed GST pull-down assays using recombinant GST-α-PIX-SH3 and GST-β-PIX-SH3 fusion proteins and lysates from COS7 cell lines expressing either *Pak3*-WT or *Pak3*-R67C variant. Quantification of PIX binding showed that PAK3-WT has a significantly higher affinity for β-PIX compared to α-PIX (β/α ratio = 8.5) (Fig. 7A). This assay also revealed that PAK3-R67C displayed a significant 3-fold increase in binding to α-PIX (Fig. 7B) and a trend toward a 1.9-fold increase in binding to β-PIX compared to PAK3-WT (Fig. 7C). To confirm this result, we performed co-immunoprecipitation assay with plasmids encoding HA-tagged PAK proteins with FLAG-tagged α-PIX and β-PIX. Quantification of the α-PIX signal, normalized to HA-PAK3 signal, showed a 33% increasing trend in PAK3-R67C binding to α-PIX compared to WT (Fig. 7D, E). We also found a tendency to increased PAK3-R67C/βPIX interactions by 21% (Fig. 7F, G). These results indicate that the R67C mutation strengthens the interaction between PAK3 and both PIX isoforms.

We then investigated whether changes in PIX binding could affect cell anchorage (Duarte et al., 2020). To this end, we induced the expression of *Pak3*-WT or R67C variants upon doxycycline treatment in COS7 cell line. Cell adhesion points were identified using anti-paxillin immunolabelling (Fig. 7H), quantified in doxycycline-treated cell lines, and compared to those in untreated cells. Expression of the WT-PAK3 protein did not change the number of adhesion points observed per cell compared to non-induced cells, as indicated by a ratio close to 1. In contrast, expression of the R67C variant significantly increased the number of paxillin-positive points per cell, indicating an increase in

focal adhesions (Fig. 7I). Since focal adhesions are essential structures that link extracellular signals to the actin cytoskeleton and play a crucial role in cell motility, and considering we have previously shown that *Pak3* variations can trigger a decrease in cell migration, we evaluated the motility of R67C-expressing cells (Duarte et al., 2020). Using the in vitro wound healing test, we induced the expression of PAK3-WT, kinase-dead or R67C variants by addition of doxycycline and we blocked proliferation with the antimitotic agent AraC. The gap relative closure was quantified 3 and 5 h after insert withdrawal. While expression of a kinase-dead form significantly reduced gap closure as previously described, expression of the R67C variation did not alter the migration rate of the cell line (Fig. 7J).

Altogether, these biochemical data, together with those previously obtained, indicate that the R67C mutation in the amino-terminal region of the protein alters kinase activity and partner affinity. These changes induce modifications in complexes involved in interactions with the extracellular matrix, without affecting cell migratory capacity, at least under the conditions tested (Combeau et al., 2012; Kreis et al., 2007; Thévenot et al., 2011).

### 3. Discussion

In this study, we report the first clinical description of the second occurrence of the most amino-terminal missense variation in PAK3. The clinical characterization of three patients from this second family confirms the previously described phenotype and further expands it, notably by revealing motor impairments and autistic features. Moreover, our phenotypic analysis of the *Pak3*-R67C knock-in mouse model confirms these findings but also highlight additional phenotypic traits, such as microcephaly. Together, the clinical and experimental data
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<figure><img src="image_28.png" /><figcaption>Fig. 6. Microcephaly of adult *Pak3-R67C* mice.</figcaption></figure>

(A) Photos of wild type (WT, on the left) or *Pak3-R67C* (R67C, 3 images on the right) perfused adult mouse brains. The scale bar is 5 mm.

(B) Mouse brain weights normalized to total body weight (n = 8 WT and 18 R67C, Student t-test, *p* = 0.0015).

(C) Whole brain volume obtained by MRI normalized to total body weight (n = 6 WT and 6 *Pak3-R67C* mice, Student t-test, *p* = 0.0495).

(D) 3D brain reconstructions based on MRI images of a WT (top images) and a R67C mouse (bottom images). From left to right: lateral views with the cortex visible (first image) or semi-transparent (second image), axial view with semi-transparent cortex (third image).

(E) Volume of distinct brain structures normalized to the mean volume in WT mice (6 WT and 6 *Pak3-R67C*; 2-way ANOVA; brain structure factor: F (3.648, 36.48) = 3.564, *p* = 0.0174; genotype factor: F (1,10) = 35.20, *p* = 0.0001; brain structure x genotype interaction: F (5, 50) = 3.564, *p* = 0.0078; post hoc analysis Benjamini, Krueger and Yekutieli: Co q = 0.0002, Ob ns, St q = 0.0301, Ve q = 0.0012, Hi q = 0.0021, Ce q = 0.0144).

provide a more comprehensive understanding of the phenotype associated with this rare variation, thereby improving our knowledge of the genotype-phenotype relationship in *PAK3*-related disorders.

Speech disorders, such as slow or inarticulate speech, sometimes associated with facial hypotonia and drooling, have been repeatedly documented in individuals carrying other PAK3 variations (Peippo et al., 2007; Rejeb et al., 2008; Horvath et al., 2018; Nagy et al., 2020; Pascolini et al., 2021). Clinical reports often fail to distinguish between a language delay due to an intellectual disability and an articulatory disorder linked to a motor problem in the orofacial area. In the patients of two families carrying the R67C variation, language disorders, and speech difficulties have been reported. For the MRX47 family, who had a neuropsychological evaluation, it seems clear that this was a language problem and not a speech problem. For the #575 family, for which data are limited, distinction was not possible but both problems may have been associated. It cannot therefore be ruled out that speech and language defects in PAK3-R67C patients have a dual origin, cognitive and muscular. Abnormalities of muscular origin could be paralleled by certain gait difficulties also described in some PAK3 patients. In addition, Pak3-R67C knock-in mice show mild motor strength deficits, despite normal motor coordination. These results collectively suggest that oro-motor difficulties, muscle weakness and cognitive impairments are defining features of PAK3-related disorders.

At the psychiatric and behavioural levels, the three patients from the

second family displayed distinct yet overlapping anomalies. Two individuals exhibited deficits in social interaction and attention, while the third presented with stereotypic behaviours and obsessive traits. Consistent with these findings, *Pak3-R67C* mice displayed subtle but significant social deficits. Specifically, they exhibited increased avoidance behaviour during social interaction with unfamiliar adult mice, despite normal interaction duration and no signs of increased aggressiveness. Notably, the *Pak3-R67C* model underscores that social dysfunction can be a key feature of *PAK3*-related disease, even in the absence of severe cognitive impairment. Furthermore, *Pak3-R67C* mice did not exhibit anxiety- or depression-like behaviours and displayed normal sensory integration, aligning with the clinical observations in R67C patients, who showed no signs of depression or anxiety, in contrast to patients bearing the W446S or the S527G variations (Horvath et al., 2018; Peippo et al., 2007). Overall, these data strongly support the pathogenicity of the R67C variation and validate the *Pak3-R67C* knock-in mouse as a relevant model for studying the psychiatric and behavioural anomalies associated with this variant.

Our previous study of *Pak3-R67C* mice demonstrated that adult males carrying the R67C variation exhibit selective deficits in long-term spatial memory and pattern separation function (Castillon et al., 2020). Here, we report an additional learning and memory deficit. Indeed, during fear conditioning tasks, *Pak3-R67C* mice displayed a mild delay in learning during the cued task but ultimately achieved memory
<figure><img src="image_29.png" /><figcaption>Fig. 7. PAK3-R67C / PIX interactions and related cellular phenotypes.</figcaption></figure>

(A) PAK3-WT protein binding to SH3 domains of α-PIX and β-PIX. Pulldown of equal amount of PAK3 were done with 10 µg of purified GST-SH3-α-PIX and GST-SH3-β-PIX and then analysed with HA antibody after capillary electrophoresis (n = 4, unpaired t-test, *p* = 0.024).

(B) PAK3-WT and -R67C protein binding to SH3 domain of α-PIX. Pulldown of equal amount of PAK3 variants were done with 10 µg of purified GST-SH3 α-PIX and then analysed with HA antibody after capillary electrophoresis (n = 4, unpaired t-test, *p* = 0.021).

(C) PAK3-WT and -R67C protein binding to SH3 domain of β-PIX. Pulldown of equal amount of PAK3 variants were done with 10 µg of purified GST-SH3 β-PIX and then analysed with HA antibody after capillary electrophoresis (n = 4, unpaired t-test, NS).

(D) α-PIX co-immunoprecipitated with PAK3: Western blot anti-HA (PAK3) or anti FLAG (α-PIX).

(E) Ratio of FLAG-α-PIX to HA-PAK3 signal (n = 7 WT and 7 R67C experiments, unpaired t-test, NS).

(F) β-PIX co-immunoprecipitated with PAK3: Western blot anti-HA (PAK3) or anti FLAG (β-PIX).

(G) Ratio of FLAG-β-PIX to HA-PAK3 signal (3 experiments for each variant, unpaired t-test, NS).

(H) Top immunofluorescent images: Paxillin signal, middle images: HA-PAK3 signal, bottom images: merge of the two signals. On the left is an example of images obtained for cells expressing PAK3-WT, on the right cells expressing PAK3-R67C. The scale bar is 20 µM.

(I) Ratio of the number of focal adhesions from induced cells normalized to non-induced cells (134 cells transfected with *Pak3*-WT and 116 cells for *Pak3*-R67C, 3 independent transfections for each genotype, Wilcoxon Mann-Whitney, *p* = 0.0020).

(J) Wound healing test. Gap relative closure for each variant normalized to its own control (non-induced cells). Three independent experiments were performed and graphics represent the mean ± SEM of relative closure 3 and 5 h after insert withdrawal (One sample Wilcoxon test, comparison to theoretical mean of 1, *p* = 0.0016, *p* = 0.0018 for WT at t3 and t5, respectively, NS for R67C at t3 and t5, *p* < 0.0001 for KD at t3 and t5).
retention comparable to WT mice. However, in the contextual task, they exhibited a learning deficit, suggesting impaired context-shock associative learning. These cognitive alterations in *Pak3-R67C* mice were mild, aligning with clinical observations of patients carrying the *PAK3-R67C* mutation (des Portes et al., 1997).

Our findings indicate that synaptic transmission is reduced at CA3-CA1 hippocampal synapses in adult *Pak3-R67C* mice. This reduction is likely not associated with presynaptic defects, as release probability at these synapses remains unchanged. Likewise, no anomalies were observed in postsynaptic structures, as evidenced by normal PSD size. However, the observed decrease in synaptic transmission may be related to a reduction in synapse number per neuron in the *stratum radiatum* of the CA1 region of the dorsal hippocampus. These results are intriguing, as existing data suggest that group I PAKs act as negative regulators of synaptic transmission. For instance, inhibition of group I PAK in the hippocampus via the expression of an autoinhibitory domain leads to increased synaptic transmission (Hayashi et al., 2004). Similarly, *Pak1/3* double KO mice exhibit enhanced synaptic transmission (Huang et al., 2011). Moreover, inhibition of the interaction between PAK3 and NCK adaptors also results in increased evoked transmission in cortical neurons (Thévenot et al., 2011). These findings suggest that the PAK3-R67C variant might confer a gain-of-function effect rather than a loss-of-function effect. Given that the PAK3-R67C variant shifts signalling toward RAC1 at the expense of CDC42, it is potentially leading to prolonged PAK3 activation, since RAC1 has a longer activated half-life compared to CDC42. Furthermore, differences between RAC1 and CDC42, such as their intracellular localization and the distribution of their respective GAPs and GEFs, may contribute to the differential activation of PAK (Aoki et al., 2005).

We found that LTP is reduced in the hippocampus of *Pak3-R67C* mice. Previous studies using *Pak3-KO* mice demonstrated L-LTP deficits using the same induction protocol and mice of a similar age as in the present study (Meng et al., 2005). In *Pak3-KO* mice, this defect was attributed to decreased CREB activity, a key transcription factor involved in late-phase LTP. As structural plasticity was not assessed in that study, it remains possible that the observed LTP deficits are associated with structural abnormalities (Boda et al., 2004; Dubos et al., 2012; Hayashi-Takagi et al., 2014). In our study, we observed a reduction in LTP induction, which depends on protein phosphorylation, receptor trafficking, and actin dynamics. Studies involving *Rac1* and *Cdc42* knockouts, both key activators of PAKs, have revealed LTP impairments and memory deficits, highlighting the significance of this pathway, possibly via PAK regulation (Haditsch et al., 2009; Kim et al., 2017). PAK3 plays a crucial role in actin cytoskeleton remodelling through its regulation of cofilin, a key protein involved in dendritic spine morphology. Additionally, PAK3 directly phosphorylates the GluA1 subunit of AMPA receptors at serine 863, promoting AMPA receptor membrane trafficking (Hussain et al., 2015). Thus, the LTP deficits observed in *Pak3-R67C* mice may stem from PAK3-mediated actin dysregulation, impaired AMPA receptor targeting, or a combination of these mechanisms. Moreover, we cannot exclude the possibility that transcription factors such as CREB are also dysregulated.

Another important observation is that adult *Pak3-R67C* mice exhibit significant microcephaly, with volumetric reductions across various brain regions, particularly in the hippocampus and cerebellum. However, microcephaly has not been reported in patients carrying the R67C mutation, neither in the first documented occurrence nor in patients from the second family (des Portes et al., 1997). These apparent conflicting observations raise the question of whether these patients might present with subtle microcephaly that has gone undetected or unreported. The criteria for defining microcephaly in humans rely on head circumference measurements, requiring values at least two standard deviations below the mean, but this is an indirect evaluation, measuring the skull, which does not reflect the exact underlying brain volume. In contrast, in mouse models, advanced imaging and volumetric analysis allow for more precise assessments of brain size, enabling the detection

of milder forms of microcephaly. In humans, brain volumetry requires the use of a dedicated software which can be applied on appropriate sequences of structural MRI but this is not performed routinely. Moreover, brain MRI is often not feasible in patients with ID and unfortunately was not available for the two families involved. Interestingly, several reports of *PAK3* missense mutations describe cases of microcephaly, including variants such as V294M, V326L, K389N/T, G424R, W428R, W446S, and Q447F (Almutairi et al., 2021; Duarte et al., 2020; Iida et al., 2019; Magini et al., 2014; Nagy et al., 2020; Pascolini et al., 2021; Peippo et al., 2007). While microcephaly is more frequently observed in severe cases, we propose that mild microcephaly may also be a characteristic feature of less severe forms of PAK3-related disease. The cellular dysfunctions induced by *PAK3* mutations could plausibly contribute to this phenotype. PAK3 is known to regulate neuronal branching, and reduced branching was observed in immature adult-born neurons in *Pak3-R67C* mice (Castillon et al., 2020; Dai et al., 2014). Additionally, PAK3 influences the cell cycle exit of neural progenitors. However, our findings of increased soma density in the *stratum pyramidale* suggest a decrease in dendritic and axonal arborization rather than an increase in cell number (Magne et al., 2021; Souopgui, 2002). Given the established roles of PAKs in dendritic spine formation and neuronal migration, and the specific involvement of PAK3 in these processes, it is plausible that the *PAK3-R67C* variant disrupts these mechanisms, ultimately contributing to reduced brain size (Boda et al., 2004; Cobos et al., 2007; Hayashi et al., 2004; Kreis et al., 2007; Viou et al., 2024). At the molecular level, our in vitro data revealing anomalies in PIX binding may provide a mechanistic explanation for these cellular defects. The PIX/PAK complex plays a critical role in multiple neuronal dynamic processes, including migration, neurite outgrowth, and spinogenesis (Santiago-Medina et al., 2013; Zhang, 2005). Disruptions in this complex due to the *PAK3-R67C* variant may therefore contribute to the observed microcephaly and other neurodevelopmental abnormalities associated with this mutation.

About the pathogenic mechanism, we demonstrated that the R67C mutation leads to several functional changes in the protein, including altered activation by GTPases, reduced or modified binding affinities with PIX and NCK partners, and a disrupted ability to form heterodimers with PAK1. These are not all-or-nothing changes, but rather shifts in the strength or efficiency of specific functions, such as kinase activity or protein-protein interactions. These effects may stem directly from structural changes caused by the mutation, or they may emerge indirectly through cascading effects. For example, the R67C mutation alters a region of the protein that normally interacts with GTPases, which can modify kinase activation and thus, in turn, may influence the auto-phosphorylation of sites critical for binding to PIX and NCK, or for dimerization with other proteins. As a result, the mutation could disrupt the fine-tuned regulation of kinase activity and PAK3 localization within the cell.

To resume, this study confirms the pathogenicity of the *PAK3-R67C* variant, as key clinical features such as deficits in memory, social interaction and motor strength/tonicity are recapitulated in the corresponding mouse model. It also identifies new phenotypic features associated with the R67C variant, observed in the model, such as microcephaly. Altogether, these results enable us to broaden and refine the clinical spectrum associated with this specific *PAK3* variation. Our work highlights alterations in synaptic transmission and plasticity that occur independently of changes in synaptic ultrastructure. Biochemical analyses reveal altered protein-protein interactions, which may underlie the observed cellular phenotypes. Importantly, this study describes in detail the first mutation-specific model, which offers clear advantages over full group I PAK inhibition experiments and over *Pak3* knock-out model, enabling the study of mechanisms specifically linked to PAK3, and more precisely to the R67C variant. It provides valuable insights into the molecular and cellular mechanisms involved, and supports the hypothesis of multiple pathogenic pathways contributing to the wide clinical variability observed in *PAK3*-related disorders.
# 4. Material & methods

## 4.1. Animal model

The Pak3-R67C mouse was previously created by our team (Castillon et al., 2020). Briefly, the strain was generated by homologous recombination after mutagenesis of the corresponding base in the recombination vector containing the second Pak3 coding exon. Mutated males and WT littermates were generated by crossing heterozygous females with WT C57Bl/6J males. Mutagenesis and mice genotype were verified by Sanger sequencing of genomic DNA and Pak3 mRNA. Expression of the mutated allele is comparable to that of the WT littermates (Castillon et al., 2020). All the mice were housed in a temperature and light-controlled colony room (12 h light/dark cycle; 21 °C, 25.5 % humidity) in groups of 2–5 per cage with food and water ad libitum. Experiments were conducted during the light phase and performed blind to the genotype. Experimental procedures were conducted in accordance with the guidelines established by the European Communities Council Directive (2010/63/EU Council Directive Decree) and the French Animal Experimentation Ethical Committee (CEEA N°59, authorization 2024-020612575357). All efforts were made to reduce the number of mice used and to minimize their suffering.

## 4.2. Magnetic resonance imaging (MRI)

4 to 6 month-old mice were imaged under isoflurane anaesthesia (induction 2 %; flow rate 2–1.8 % in 20 % O2, 80 % air) controlled on the basis of respiratory parameters. The body temperature was maintained at 37 °C using a heated mattress. MRI measurements were performed on a 7-T horizontal bore magnet (Oxford, UK) driven by Paravision (Bruker®), Wissembourg, France), equipped with a 300mT/m actively shielded gradient device (internal diameter [ID] = 90 mm, Bruker®). For MRI examination, the animal's head was introduced in a “bird-cage” 1-H coil (ID = 20 mm). The positioning and shimming processes were followed by 3D-TurboRARE (TR/TE eff = 1500/40 ms (repetition time/effective echo time); 2 averages; 20\*20\*20 mm field of view; rare factor = 8; 256\*192\*40 matrix; voxel size, after reconstruction = 78\*78\*250 mm³). The total time spent by the mouse in the magnet never exceeded 1 h. After each experiment, mice were recovered from anaesthesia and returned to their home cages with free access to food and water. Areas and volumes were estimated in both the coronal and sagittal MRI planes using the AMIRA software (TGS Inc., San Diego, CA). Analyses were performed as previously described (Gonzalez et al., 2023) with some modifications: the mouse brain atlas and reference database were used as initial guides and the brain was first subdivided into 12 main regions of interest (ROI). (Ma et al., 2005; Paxinos and Franklin, 2019).

## 4.3. Electrophysiological recordings

### 4.3.1. Slice preparation

Male mice aged 2 to 4 months old were used for field recordings. Mice were euthanized using cervical dislocation and then swiftly decapitated. Hippocampus extraction was made on ice and placed on an agar-agar block in ice-cold artificial cerebrospinal fluid (aCSF). The composition of the aCSF was as follows (in mM): 119 NaCl, 2.5 KCl, 1.3 MgSO4, 2.5 CaCl2, 1 NaH2PO4, 26 NaHCO3, and 11 glucose, equilibrated with 95 % O2 and 5 % CO2. Transverse acute hippocampal slices (400 µm thick) were prepared using a Leica T 1200S vibratome with a platinum coated blade (Feather NEW Hi-STAINLESS). Slices were maintained at 33 °C in a storage chamber perfused with aCSF during 1 h before recordings. After the recovery period, a slice was put on a Poly-L-Lysine coated cover slip and put in a chamber under a microscope (AxioScope Zeiss). The chamber was perfused with oxygenated aCSF at a flow rate of 2.0 ml/min. The temperature of the perfusing aCSF was maintained between 30 and 33 °C using the Automatic Temperature

Controller (TC-324B, Warner Instruments).

### 4.3.2. Data acquisition

The stimulus electrode was placed in a 4-5 MΩ borosilicate glass capillary (1.5 OD × 0.86 ID × 100 L - Phymep) stretched with a pipette puller (P-97 Sutter instruments) and filled with 1 M NaCl. Recording electrode was placed in a 1 MΩ capillary (1.5 OD × 0.86 ID × 100 L - Phymep) filled with aCSF. Extracellular field excitatory postsynaptic potentials (fEPSPs) were recorded in the *Stratum Radiatum* of the CA1 using, as follow: an Axopatch-700 A amplifier (Axon instruments), a HEKA C-1600 acquisition system interface, a Hum Bug 50/60 Hz Noise Eliminator and a data acquisition software (Elphy). All stimulations were set to have a 0.4 ms width (using a DS2A voltage stimulator - Mk. II). Stimuli were delivered to the Schaffer collateral axons in the CA1 region and fEPSP were recorded in the *Stratum Radiatum*. The electrodes were moved using a micromanipulator (PCS-5000 Burleigh).

### 4.3.3. Input/output protocol

Input/output protocols were conducted with stimuli ranging from 0 to 70 V, using a 5 V increment from 0 to 30 V and a 10 V increment from 30 to 70 V. Three stimulations were delivered at each intensity, and the mean response for each intensity was calculated.

### 4.3.4. Paired-pulse

The intensity that elicited 30 % of the maximal fEPSP slope, as determined during the input/output protocol, was used during paired-pulse protocols. Two pulses of equal intensity were delivered with inter-pulse intervals of either 50, 100, 150, or 200 ms.

### 4.3.5. Long-term potentiation

The intensity that elicited 30 % of the maximal fEPSP amplitude, as determined during the input/output protocol, was also used to establish a baseline for the LTP protocol. fEPSP were measured every 30s during the 2 h-LTP, with the exception of the stimulus train delivery. fEPSPs were recorded until a 20 min stable baseline was obtained. LTP was then induced by delivering 3 stimulus trains of 100 Hz for 1 s, each train separated by a 10s interval.

## 4.4. Electron microscopy

For electron microscopy, 4 to 6 month-old mice were used (n = 8 WT, 7 R67C). Mice received an overdose of sodium pentobarbital (150 mg/kg) and transcardiac perfusion was performed with PB containing 4 % paraformaldehyde and 0.1 % glutaraldehyde. After 1 h at 4 °C, the brains were extracted from the skull and postfixed in 4 % paraformaldehyde overnight at 4 °C. The brains were then stored in PBS containing 0.4 % sodium azide at 4 °C. 200 µm-thick frontal sections were cut in PBS with a vibratome. For each brain, a section located in the middle part of the dorsal hippocampus was collected in PBS. The sections were transferred into an osmium solution for one hour. After several washes, they were stained “en bloc” with an uranyl acetate solution for one hour. After washes, the samples were dehydrated in graded ethanol followed by acetone. They were incubated in progressive concentrated epoxy resin before being embedded in pure resin. Three semithin sections (0.5 µm-thick) 15 µm apart per case were stained with 0.5 % toluidine blue. Three measurements of the height of the *stratum radiatum* were performed on each section (Fig. 3A). The number of neuronal nuclei profiles in the pyramidal layer was counted in a 310 µm-width band of the *stratum pyramidale* on the three semithin sections per case (Fig. 3C). Ultrathin sections (70 nm-thick) were collected on 200 mesh copper grids for electron microscopy analysis. The sections were contrasted by incubation with lead citrate for 8 min, and were analysed with a Hitachi HT 7700 (Elexience, Verrieres le Buisson, France) electron microscope. The number of synaptic profiles on spines in the *stratum radiatum* was counted in a 53 µm² at 50–100 µm from the pyramidal layer. A mean of 30 measurements were performed in one ultrathin
section (every two fields in one square of the 200-mesh grid). The length of 100 postsynaptic densities (PSDs) per mouse were measured on the same photomicrographs.

## 4.5. Behavioural testing

Behavioural tests were all conducted on male mice aged from 2.5 to 4 months. Unless stated otherwise, acquisition was done using ANY-maze software (Stoelting, USA).

### 4.5.1. Traction test

Each mouse's forepaws was placed on a thin horizontal wire (1.5 mm in diameter), positioned 35 cm above the surface of a table. The time taken for the mouse to bring at least one hind paw up to grip the wire was recorded as a successful traction. Each mouse underwent three trials, with a 10-min inter-trial interval. Each trial had a maximum duration of 60s. A score was attributed according to the mouse performance: 0 – falls without traction, 1 – attempts a traction, 2 – one hind paw grips the wire, 3 – both hind paws on the wire, 4 – all paws on the wire and tail around the wire, 5 – mouse escapes sideways.

### 4.5.2. Inverted grid test

Each mouse is placed on a cage wire grid 35 cm above an empty box with clean litter. After slowly inverting the grid upside-down to 180°, the ability of the mouse to maintain a grip onto the grid was monitored (grip latency); with a maximum score of 120 s being given if the animal did not fall.

### 4.5.3. Rotarod test

Each mouse is placed on a rotating cylinder set at a constant speed of 0, 4, or 16 rpm (rpm) for up to 180 s. The latency to fall is recorded, and if the mouse remains on the cylinder for the full 180 s, this maximum time is noted. For each rotation speed, three consecutive training sessions are conducted, separated by 10-min intervals. Additionally, for the accelerated rotarod test (ramping from 4 to 40 rpm over 300 s), mice undergo five training sessions per day, each separated by 10-min intervals, for three consecutive days.

### 4.5.4. Plus-maze test

The maze consisted of two opposing arms enclosed by high walls (20 × 8 × 25 cm), two open arms (20 × 8 cm), and a central area (8 × 8 cm) arranged in a plus shape, elevated 65 cm above the floor. Lighting was set at 100 lx for the center and the open arms and 30 lx in the enclosed arms. Mice were individually placed in the center of the maze, facing an open arm. The number of entries and the time spent in the open and enclosed arms were recorded over a 5-min period.

### 4.5.5. Tail suspension test

This test was used to study resignation behaviour. Each mouse is suspended by its tail from a wire 35 cm above the ground during two sessions separated by 24 h, each lasting 6 min. The latency to the first moment of immobility (superior to 2 s) and the duration of immobility are manually quantified.

### 4.5.6. Forced-swim test

Two 5-min sessions are conducted, separated by a 24-h interval. During each session, the mouse is placed in a beaker (11 cm in diameter, 25 cm in height) filled with water (18 cm deep) at approximately 25 °C. Upon being placed in the water, the mouse typically begins by struggling, attempting to climb up the beaker walls in vain ("climbing"). Gradually, other observable responses can be quantified, reflecting the mouse's progressive resignation to the inescapable situation: immobility and floating, characterized by small movements of the hind limbs that allow the mouse to keep its head above the water without moving around.

### 4.5.7. Startle and prepulse inhibition

The experimental setup consists of a square chamber with three black walls and a transparent front door (25 cm per side). The animals are placed in a plexiglass cylinder attached to a grid bar on the floor (5 × 10 × 5.5 cm), allowing some movement without restraining them. Movements and startle responses are detected by a piezoelectric accelerometer in contact with the grid. A speaker, positioned 20 cm above the chamber, emits a background white noise of 65 dB and delivers stimuli of varying intensities and frequencies. The apparatus is placed inside a dark, ventilated, soundproof enclosure (67 × 53 × 55 cm) on an anti-vibration table. The presentation of stimuli is controlled by the Startle software (PanLab, Spain), which digitizes, corrects, and records the accelerometer's responses. The system is calibrated at 10 kHz using a sound level meter, and the generated sounds are sinusoidal signals with a smooth rise and fall (5 ms each) to ensure accurate reproduction without artifacts. The startle response test consisted of determining the intensities of auditory stimuli capable of triggering a startle response in mice. The session consisted of six blocks of nine trials, during which pulses of varying intensities (5, 10, 20, 25, 30, 40, 45, 50 dB above background noise) were presented in a pseudo-random order at intervals of 10, 15, or 20 s. The second experiment focused on studying the inhibition of the startle response by a prepulse. The session included 6 blocks of 5 trials, where a non-startling pre-pulse of varying intensity (0, 3, 6, 9, 12 dB above background noise, 10 kHz, 20 ms) was presented 100 ms before the startle-inducing pulse (55 dB above background noise). The pre-pulses were presented in a pseudo-random order. To evaluate the percentage of startle inhibition by a pre-pulse, the startle amplitude was normalized for each mouse to its response amplitude when presented with a pre-pulse of 0 dB above background noise, which induces no inhibition, resulting in a maximum startle response (% of maximum response). A startle response was defined as having a latency of less than 120 ms and an amplitude greater than 10 arbitrary units (A. U.).

### 4.5.8. Auditory-cued fear conditioning

The conditioning procedure was performed using the StartFear system (Panlab, Barcelona, Spain) in a chamber (25 × 25 × 25 cm) with three black methacrylate walls and a transparent front door. The chamber had a grid floor connected to a shock scrambler (UCS) and a speaker on the ceiling to deliver audible tones (CS), with background white noise set at 60 dB. The apparatus was placed on a high-sensitivity weight transducer system for tracking mouse movement, all within a ventilated, soundproof enclosure on an anti-vibration table. On the first day (habituation), mice were placed in the chamber for 5 min of free exploration. The next day (acquisition), following a 3-min acclimatization, mice were exposed to five CS-UCS trials, with pseudorandom intervals of either 60s, 120 s, or 180 s. Each trial consisted of a 30-s tone (80 dB, 10 kHz), followed by a 2-s, 0.4 mA, foot shock. After 24 h (retention), the mice were placed in a novel context (white walls and a different textured floor) for 3 min of baseline observation, followed by five CS presentations without shocks, with ITIs of 60s, 90s, and 120 s. Movements were continuously monitored by an accelerometer to measure periods of immobility ("freezing"), which were analysed using Freezing software (Panlab) and normalized as a percentage of the total session to quantify fear responses and associative learning.

### 4.5.9. Contextual fear conditioning

The conditioning setup consisted of a square testing box (30 × 30 cm) with a grid floor for delivering electric foot shocks (unconditioned stimuli) and clear Plexiglass walls (45 cm high) without a ceiling for full observation and video tracking. The experiment was conducted under moderate lighting (150 lx). On the first day (acquisition phase), each mouse was allowed to explore the box freely for 2 min, followed by three 0.4 mA foot shocks (2 s each) at 2-min intervals. After the last shock, the mouse remained in the box for an additional 2 min. In the recall phase, occurring 24, 48, 72 and 96 h later, each mouse was returned to the
same box for 20-min sessions without receiving any shocks. Freezing behaviour, indicating fear response and memory retention, was tracked and analysed using AnyMaze software.

### 4.5.10. Spatial object recognition

The testing box is a circular open field (48 cm in diameter and 30 cm in height) with the floor covered in sawdust (lighting: 50 lx at the center). On the morning of the first day (open field familiarization), each mouse is gently placed in the center of the apparatus for 10 min. In the afternoon (familiarization with object exploration), the mice are again placed in the center for 10 min and can explore 4 'non-attractive' objects (2.5 cm in diameter and 1 cm in height). On the second day (acquisition), the mice undergo 3 successive 5-min trials, spaced 5 min apart, where they can explore 3 different objects "Legos" (about 7 cm in height and 4.5 cm in width) placed at the center of 3 quadrants, forming a triangle. An observation zone about 10 cm in diameter is marked around each object. The time spent exploring each object is quantified (nose in contact with the object or oriented toward the object within the observation zone). The next day (restitution), the mouse undergoes a single 5-min session during which it can explore the 3 objects from the previous day, but one of the objects has been moved to a novel, previously empty, location. Memory performance is quantified by the ratio of the time spent exploring the displaced object to the total exploration time of the 3 objects, multiplied by 100; the chance threshold is 33.33 % for each object.

### 4.5.11. Social interaction test

The Social Interaction Test -SIT- is designed to assess social flexibility and adaptability to a novel conspecific as animals are free to interaction and to organize social sequences (de Chaumont et al., 2012; Granon et al., 2003). It takes place in a novel environment (transparent plexiglas box 1 25 X L 50 X H 45) containing a handful of clean bedding, located in a novel experimental room with luminosity set at 100 Lux. The test mouse, previously isolated for 3 weeks at adult age, is placed in the box for 30 min before a novel conspecific of same sex and age (hereby called the visitor mouse, always maintained in a group of 4 cage) was introduced. The two animals have not been in contact before and they are free to interact or not. The social interaction is recorded for 8 min and all behavioural parameters are analysed off line using Solomon coder. We analysed the number and duration of social contact (either oro-oral,orgenital or any body touch) between the two individuals, the number and duration of social follow behaviour (i.e. pursuit), that we previously showed to be associated with dominance behaviour, the number of time the test mouse places its forepaw on the head or the back of the visitor mouse, the number of tail rattling and bites and the number of self grooming episodes (Coura et al., 2013).

## 4.6. Biochemistry and cell imaging

### 4.6.1. Stability

PAK3 expression and stability was assessed upon cycloheximide treatment to inhibit protein synthesis. COS7 cells were transfected with pcDNA3 construction expressing HA-tagged proteins as previously described (Kreis et al., 2007). Two day after transfection, cells are split in three dishes and cycloheximide (10 mg/ml) was added and cells were lysed immediately (t0), 16 h, and 36 h after. Quantitative Western blots were done with HA antibodies. Expression was calculated relative to the t0 value. Stability was also evaluated using stable COS7 cell line expressing PAK3-R67C variant upon induction with doxycycline (0.2 µg/ml) as previously described (Duarte et al., 2020). Briefly, after 48 h of PAK3-R67C and WT expression, protein synthesis was stopped by doxycycline withdrawal, cells were lysed immediately and at 24 h and decrease of PAK3 protein level was monitored by western blot using anti-HA antibodies and with anti-β-tubulin as loading control.

### 4.6.2. Plasmid cloning of pGEX-SH3-α-PIX and pGEX-SH3-β-PIX

The SH3 domains of α-PIX and β-PIX were amplified by PCR from FLAG-α-PIX or FLAG-β-PIX plasmids using the following primers: forward 5'-ctgGGATCCAacaatcaactggtagtaagagcaaagtttaact-3' and reverse 5'-gcggccgCTCGAGttagccttgacctcgcgcac-3', containing BamHI and XhoI restriction sites (capital letters), respectively. The ~205 bp PCR products were digested with BamHI and XhoI, and DpnI (New England Biolabs). In parallel, the pGEX-6P3 vector was digested with BamHI and XhoI. Digested inserts were ligated into the linearized vector and transformed into Top10 competent E. coli cells. Positive clones were selected on antibiotic-containing plates, screened by colony PCR, and verified by Sanger sequencing. The recombinant GST fusion proteins (GST-β-PIX-SH3 and GST-α-PIX-SH3) were expressed in E. coli -BL21 by adding IPTG, and purified using magnetic glutathione beads (Thermo Fisher Scientific). Protein concentrations were determined using Coomassie blue staining of the polyacrylamide gel, with BSA as standard range.

### 4.6.3. PIX interaction

Coding sequence for PAK3-R67C was introduced in the pHAGE vector and stable COS7-Lv-PAK3-R67C were established as described for other variants (Duarte et al., 2020). For pulldown assay, established COS7 cell lines were treated with doxycycline in order to express HA-PAK3-WT or -R67C variants, during 24 to 48 h, and then where lysed in NP-40 lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 % NP-40, supplemented with protease and phosphatase inhibitors). Expression of the two variants were quantified by HA-western blot on the same membrane, and equal amount of signal were used for the assay. Equal amounts of GST-PIX-SH3 magnetic beads (10 µg) were incubated with increasing amounts of cell lysates for 2 h at 4 °C with gentle rotation. Beads were then washed four times with lysis buffer and were suspended in 15 µL of NP-40 lysis buffer. Samples were analysed using the WES capillary Western blot system (Bio-Techne) according to the manufacturer's instructions, with HA antibodies (12CA5, Roche).

Co-immunoprecipitation assays were realized as previously described for others PAK3 variants (Duarte et al., 2020). Briefly, cells were co-transfected with the two plasmids of interest with Lipofectamine. Transfected cells were lysed 36 h later with a modified Robert's lysis buffer supplemented with protease and phosphatase inhibitors mixture. After 15 min on ice, lysates were cleared by centrifugation at 10,000 g for 15 min. A Bradford protein assay (Biorad) was then performed to measure the protein concentration. HA-tagged PAK3 proteins were immunoprecipitated overnight at 4 °C using monoclonal anti-HA agarose antibody (Sigma-Aldrich, #A2095). Protein samples were separated by 10 % SDS-PAGE. Immunoblot analysis were performed using anti-HA (Cell Signalling Technology, #2367), anti-FLAG (Sigma, #F7425) primary antibodies. Membranes were then incubated using anti-mouse (Biorad, #172-1011) peroxidase-linked secondary antibodies. Membranes were then revealed with Luminata Crescendo (Millipore). Quantification was assessed using a CCD camera (Fusion FX imaging system, Vilber Lourmat) and analysis was performed with ImageJ software. Amounts of co-immunoprecipitated FLAG-PIX proteins, detected and quantified by western blot were expressed relatively to HA-immunoprecipitated PAK3 proteins.

### 4.6.4. Analysis of focal adhesions

COS7-Lv-PAK3 cells were plated at low density on glass coverslips and protein expression was induced with doxycycline during 24 h in half conditions. Cells were fixed with 4 % p-formaldehyde for 15 min, washed with PBS and permeabilised with 0.1 % Triton X-100 and 0.1 % Tween 20 for 15 min at room temperature, then blocked with 0.3 % bovine serum albumin (BSA) for 2 h at room temperature before an incubation in 0.1 % Tween 20, 0.1 % BSA with anti-paxillin (Millipore/ Upstate, #05-417) and anti-HA (Roche, #1867423) antibodies overnight at 4 °C. Cells were then washed and incubated for 2 h at room temperature in 0.1 % Tween 20, 0.1 % BSA with Alexa-568 anti-rat
(Molecular Probes, #A11077) and Alexa-488 anti-mouse (Molecular Probes, #A21121) antibodies to label adhesion points and PAK3. Slips were mounted on slides with Fluoromount + Dapi (eBioscience, #00-4959-52), and cells were imaged using a Leica DMI6000B microscope. Paxillin-positive dots corresponding to adhesion points were quantified for each mutant cell line with or without doxycycline induction. Each quantification was then normalized to its own non-induced control. Illustrating images were obtained with a confocal microscope LSM700 (Zeiss, Germany).

### 4.6.5. Analysis of COS7 cell migration by wound healing assay

COS7-Lv-PAK3 cells were plated at 7.10⁵ cells/ml as recommended by Ibidi using a culture insert with two wells separated by a 500 µm gap (Ibidi GmbH, #81176). One day after, 50 µM of Cytosine Arabinoside (AraC) was added to each well and 2 µg/ml of doxycycline to half conditions. 24 h later, insert was removed, and wells were washed twice with PBS and a fresh complete growth medium with AraC and doxycycline (in induced wells) was added. Pictures were then taken using a Leica DMI6000B microscope at 0, 3 and 5 h after insert withdrawal. Migration analysis was performed with ImageJ software: each induced cell line migration was normalized and compared to its non-induced control.

### 4.7. Statistical analysis

Normality was tested with the Kolmogorov-Smirnov test and variance homogeneity was verified using a Levene test of homoscedasticity. When data followed a normal distribution and when variance were not significantly heterogeneous, we used a Student t-test to compare 1 factor between 2 samples (WT vs R67C). When data did not follow a normal distribution or variance homoscedasticity, we used the Mann-Whitney 2-tailed nonparametric test. For multiple comparison, 2-way ANOVA was used. For correlation, simple linear regression with Pearson's correlation was used. Statistics were performed using GraphPad Prism 8.4.3 (San Diego, California, USA). Each value is expressed as means with SEM. P-values inferior to 0.05 were considered significant.

### CRediT authorship contribution statement

**Manon Dobrigna:** Writing – review & editing, Writing – original draft, Resources, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization.
**Sandrine Poëa-Guyon:** Writing – review & editing, Writing – original draft, Supervision, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization.
**Mathieu Legras:** Writing – review & editing, Investigation, Formal analysis.
**Delphine Le Verger:** Investigation.
**Kevin Duarte:** Investigation.
**Catherine Sébrie:** Writing – original draft, Resources, Investigation, Formal analysis.
**Roseline Poirier:** Writing – review & editing, Writing – original draft, Supervision, Resources, Methodology, Investigation, Formal analysis, Conceptualization.
**Sylvie Granon:** Writing – original draft, Supervision, Methodology, Investigation, Formal analysis, Conceptualization.
**Serge Marty:** Writing – original draft, Resources, Investigation, Formal analysis, Conceptualization.
**Annick Toutain:** Writing – review & editing, Writing – original draft, Resources, Formal analysis.
**Glenn Dallérac:** Writing – original draft, Supervision, Methodology, Funding acquisition, Formal analysis, Conceptualization.
**Cyrille Vaillend:** Writing – review & editing, Writing – original draft, Supervision, Project administration, Methodology, Funding acquisition, Formal analysis, Data curation, Conceptualization.
**Jean-Vianney Barnier:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

### Funding

This study was funded by Centre National de la Recherche Scientifique (CNRS), France; Université Paris-Saclay, France, and grants from Agence Nationale de la Recherche (ANR), France: ANR-21-CE17-0053 ID-GePhe-PAK and ANR-MRGENES; from the Fondation Jérôme Lejeune 2022-#2160, the Fondation Maladies Rares, la Fédération pour la Recherche sur le Cerveau (FRC), the Association Xtraordinaire, the NeurATRIS ANR-11-INBS-0011, of the French Investissements d'Avenir Program run by the Agence Nationale pour la Recherche. M.D. and K.D. were funded with PhD fellowships from the French Ministry of Research (Doctoral School Biosigne from Paris-Saclay University and the Graduate School Life Sciences and Health).

### Declaration of competing interest

The authors declare no competing or financial interests.

All authors have read and agreed to the published version of the manuscript.

During the preparation of this work the authors used ChatGPT in order to improve the readability and language of the manuscript. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the published article.

### Acknowledgements

We thank Marie-Christine Birling for the knock-in strategy (Institut Clinique de la Souris, Illkirch) and the Central Transgenic Core facility for generating the mouse Pak3-R67C transgenic line. We thank people of the animal facility of the Institute of Neuroscience Paris-Saclay for assistance in animal breeding and general maintenance. We also thank the NeuroPICT imaging facility of the institute. We thank Dr. Lucy Raymond (Cambridge Institute for Medical Research) for providing unpublished data about clinical cases. We also thank Frédéric Laumonnier for constructive feedback.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.nbd.2025.107188.

### Data availability

Data will be made available on request.

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