Fine-Tuning the PI3K/Akt Signaling Pathway Intensity by Sex and Genotype-Load: Sex-Dependent Homozygotic Threshold for Somatic Growth but Feminization of Anxious Phenotype in Middle-Aged PDK1 K465E Knock-In and Heterozygous Mice

According to the Research Domain Criteria (RDoC), phenotypic differences among disorders may be explained by variations in the nature and degree of neural circuitry disruptions and/or dysfunctions modulated by several biological and environmental factors. We recently demonstrated the in vivo behavioral translation of tweaking the PI3K/Akt signaling, an essential pathway for regulating cellular processes and physiology, and its modulation through aging. Here we describe, for the first time, the in vivo behavioral impact of the sex and genetic-load tweaking this pathway. The anxiety-like phenotypes of 61 mature (11–14-month-old) male and female PDK1 K465E knock-in, heterozygous, and WT mice were studied. Forced (open-field) anxiogenic environmental conditions were sensitive to detect sex and genetic-load differences at middle age. Despite similar neophobia and horizontal activity among the six groups, females exhibited faster ethograms than males, with increased thigmotaxis, increased wall and bizarre rearing. Genotype-load unveiled increased anxiety in males, resembling female performances. The performance of mutants in naturalistic conditions (marble test) was normal. Homozygotic-load was needed for reduced somatic growth only in males. Factor interactions indicated the complex interplay in the elicitation of different negative valence system’s items and the fine-tuning of PI3K/Akt signaling pathway intensity by genotype-load and sex.


Introduction
The expression of psychiatric symptoms such as anxiety across lifespan still needs important research efforts to dissect and understand their modulation's biological and environmental basis [1]. Here, the new understanding of psychopathology in terms of dysregulation and dysfunction in essential behavioral features through neurobiology and behavioral neuroscience can provide a promising research scenario [2,3]. In this new conceptualization, fear, aggression, and distress are three draft constructs within the negative valence system (NVS), one of the five domains in the NIMH's Research Domain Criteria (RDoC) matrix [2,4]. This RDoC matrix comprises the interplay between behavioral dimensions or functional constructs inspected by seven different 'units of analysis', namely, genetic and molecular basis, cells and neuronal circuits, physiology of the phenotypes, behavior, and self-report [3]. The molecular genetic basis of NVS phenotypes is considered to be in its infancy, yet with few candidate genes nominated for anxiety disorders [5]. In this context, basic research on signal transduction provides an advanced close examination Therefore, the present study aimed to explore further the contribution of sex and genetic load in the expression of the somatic and anxious-like behavioral phenotype of the PDK1-K465E PH-domain knock-in mice.

Generation of PDK1 K465E/K465E Mice and Genotyping Analysis
The generation and genotyping of the PDK1 K465E/K465E knock-in mice expressing the single-amino-acid substitution of lysine 465 to glutamic acid in the PDK1 PH domain were described previously [17]. The mice were subjected to PCR genotyping of genomic DNA isolated from ear biopsy using primers K465E F (5 -GGG TGA AGC ATG GAA TCT GTG TCT T) and K465E R (5 -GCC AGG ATA CCT AAG AGT ACC TAG AA). PCR amplification resulted in a 196-bp product from the wild-type allele and a 236-bp product from the targeted allele.
Mice were maintained at the Animal House Facility of the Universitat de Lleida under standard husbandry conditions (housed three to four per cage in Macrolon cages, 35 × 35 × 25 cm, with food and water ad libitum, 22 ± 2 • C, a 12 h light: dark cycle and relative humidity 50-60%). Behavioral assessments and data analysis were performed blind to the experiment, in a counterbalanced manner, in the light cycle, from 9:00 to 13:00 h. All procedures were in accordance with Spanish legislation on 'Protection of Animals Used for Experimental and Other Scientific Purposes' and the EU Directive (2010/63/UE) on this subject. The study complies with the ARRIVE guidelines developed by the NC3Rs and aims to reduce the number of animals used [41].

Behavioral Assessments
Animals were behaviorally assessed for NVS in the open field [42] and the marbleburying tests [43], two unconditioned tests differing in their anxiogenic conditions. A graphical abstract, also including the conclusions, illustrates the methodological setting and procedures ( Figure 1).
As measured by body weight, somatic growth was recorded on Day 0 prior to the behavioral battery of tests to monitor possible confounding factors. Lack of sensorimotor problems was already described in these animals in the precedent work [36].
Day 0. Somatic Growth/Bodyweight. Bodyweight was used to measure the somatic growth and physical condition/health status of animals. Day 1. Open field test (OF). Animals were individually placed in the center of an illuminated (20 lux) open field (homemade woodwork, white box, 55 cm × 55 cm × 25 cm) and observed for 5 min. First, the ethogram of action programs (sequence of behavioral events) was analyzed. Thus, the duration of freezing behavior (latM, latency of movement) and the latency of the behavioral events that follow it were recorded: leaving the central square (latC), reaching the periphery (thigmotaxis) (latP) and performing first wall rearing (latR). Second, the time course and total levels of exploratory activity were measured as horizontal (C, number of crossings) and vertical (Rw, rearing with wall support) locomotor activity. Finally, as previously described [44] we evaluated the presence of bizarre behaviors assessed through the number of stereotyped rearings without wall support (Rc).
Day 2. Marble-Burying Test (MB). The procedure used was as previously described [45]. The mice were placed individually facing the wall in a standard home cage with six glass marbles (1 cm × 1 cm × 1 cm) on a 5-cm-thick layer of clean wood cuttings. The marbles were spaced in three rows of two marbles per row in one half of the cage. The mice were left in the cage with marbles for a 30-min period. The test was terminated by removing the mice. The number of marbles that were buried, changed position (partially buried or turned), and were left intact (I) were measured. Day 2. Marble-Burying Test (MB). The procedure used was as previously described [45]. The mice were placed individually facing the wall in a standard home cage with six glass marbles (1 cm × 1 cm × 1 cm) on a 5-cm-thick layer of clean wood cuttings. The marbles were spaced in three rows of two marbles per row in one half of the cage. The mice were left in the cage with marbles for a 30-min period. The test was terminated by removing the mice. The number of marbles that were buried, changed position (partially buried or turned), and were left intact (I) were measured.

Statistics
Statistical analyses were performed using SPSS 15.0 software. All data are presented as mean ± SEM, and are illustrated as bars that illustrate the mean values in each group segregated by genotype and/or sex, as indicated in the legends. To evaluate the effects of the genotype (G) and sex (S) group, a 3 × 2 factorial analysis design was applied. Differences were studied through Multivariate General Lineal model analysis, followed by post hoc Sidak test comparisons when it was possible. For categorical variables, the Fisher's exact test was used. Graphics were made with GraphPad Prims 6, and p-value < 0.05 was considered statistically significant.

Statistics
Statistical analyses were performed using SPSS 15.0 software. All data are presented as mean ± SEM, and are illustrated as bars that illustrate the mean values in each group segregated by genotype and/or sex, as indicated in the legends. To evaluate the effects of the genotype (G) and sex (S) group, a 3 × 2 factorial analysis design was applied. Differences were studied through Multivariate General Lineal model analysis, followed by post hoc Sidak test comparisons when it was possible. For categorical variables, the Fisher's exact test was used. Graphics were made with GraphPad Prims 6, and p-value < 0.05 was considered statistically significant.
Bizarre behavior (Rearings in the center): No main but interaction effects were also found in the rearings performed in the center of the apparatus ( Figure 4C, G × S, F(2,55) = 3.360; p = 0.043). Post hoc multiple comparison analysis showed homozygote mutant females performed less than homozygote mutant males in minutes 2 (s, p = 0.034), 3 (s, p = 0.048), 5 (s, p = 0.018), resulting in a total lower vertical rearing activity in the center ( Figure 4C

Marble Burying Test
The qualitative (three levels of interaction) and quantitative (number) analysis of the marble-burying test did not show any statistically significant effect and/or differences between groups (see Figure 5). However, in contrast to the standard quantitative evaluation Vertical behavior-Latency of rearing showed a genotype main effect ( Figure 3C, G, F (1,55) = 3.675; p = 0.032), where both PDK1+/− and PDK1 −/− genotypes performed rearing earlier than WT ( Figure 3C, *, p = 0.035 and *, p = 0.040, respectively). Post hoc multicomparison analysis also showed that latency of rearing was shorter in female WT as compared to males ( Figure 3C, s, p = 0.031) and that both PDK1+/− and PDK1−/− males performed rearing earlier than WT males ( Figure 3C, g, p = 0.016 and gg, p = 0.008, respectively). As shown in Figure 4, the min-by-min analysis of the temporal course of horizontal locomotor activity indicated similar habituation curves in the three genotypes. In the last minute of the test, the female sex performed less activity than males ( Figure 4A, S, F (1,55) = 5.763; p = 0.020). Post hoc multiple comparison analysis indicated that this sex effect was mostly due to the sexual dimorphism of WT mice in minute 5 ( Figure 4A, s, p = 0.043).

Marble Burying Test
The qualitative (three levels of interaction) and quantitative (number) analysis of the marble-burying test did not show any statistically significant effect and/or differences between groups (see Figure 5). However, in contrast to the standard quantitative evaluation protocol, the consideration of several levels of interaction with small objects enabled us to uncover the predominant behaviors. That is, in the three strains, the most common behavioral interaction did not result in the complete burying of the marbles but their change of position (turned or partially buried).

Discussion
In the present work, we describe for the first time the in vivo effects of the PDK1 mutation in the PH domain depending on age and genetic load. Mature (11-14 months o

Discussion
In the present work, we describe for the first time the in vivo effects of the PDK1 mutation in the PH domain depending on age and genetic load. Mature (11-14 months of age) male and female PDK1 −/− and PDK1 +/− PH-domain knock-in mice were studied and compared to age-and sex-matched WT mice with normal aging. The results unveil the fine-tuning of the signaling pathways by sex and genetic load, with a feminization of the behavioral profiles.

Homozygous-Load of the Mutant Gene Is Needed for the Reduced Somatic Growth Effects Only in Mutant Males
As previously described [33,35,36], somatic growth, measured through bodyweight, was found reduced in both male and female PDK1 −/− mice. It is known that homozygous male and female PDK1 −/− are −35% smaller from birth than WT littermates [33]. In that precedent work, magnetic resonance imaging-obtained images or physical sections of fixed organs using the Cavalieri method described reduced brain volume (−20%) but also of metabolic (liver), immune (spleen, −20%), male gonadal (testis, −50%) organs and a slight reduction in kidney size (albeit did not reach statistical significance) in PDK1 −/− mice compared to littermates [33]. Disector principle, a quantitative and unbiased stereological approach to estimate cell volume, also indicated that a reduction in organ volume translated into a reduction of cell size. Thus, in the case of adrenal glands of PDK1 −/− animals, a −40% reduction in the relative cell size of zona fasciculata cells was found compared to littermates. Interestingly, the present report shows that heterozygosis was enough to sustain normal weight. However, further analysis segregated by sex unveiled that the half genetic load of wild type PDK1 could guarantee normal somatic growth only in males since heterozygous females were already sensitive to this somatic effect of PDK1/Akt signaling pathway. We hypothesize that this sex-dependent modulation of size translates into the previously reported organ sizes, glucose resistance, hyperinsulinemia, and insulin resistance [33], and future experiments are needed to study it further.

Similar Neophobia and Locomotion, but Increased Anxiety-Like Phenotype in Mutant Females and Feminized Anxious-Like Phenotype of Mutant Males
In the PDK1 −/− mice, the cortical and hippocampal deficits in the PDK1/Akt signaling [34] and the anxious-like phenotype [36] are found attenuated at 11-14 months of age as compared to young adulthood. In the present work, we provide further evidence of a fine-tuning modulation of this signaling pathway by sex and genetic load. Neophobia, an amygdala-dependent immediate fear response to novelty, was similar among the six groups of animals. However, after that, mutant females exhibited a coping with stress strategy characterized by shorter latencies to develop the ethogram, thigmotaxis, increased wall rearing, and presence of bizarre rearings. As previously described, according to their temporal (repetitive/stereotyped or not) and spacial (horizontal/vertical) features, behaviors apparently without a purpose but considered coping-with-stress strategies can be classified as stereotyped stretching, stereotyped rearing, backward movement, and jumping [44]. These disrupted behaviors are scarcely observed in young animals and still difficult to record at middle age, as shown here by the low number in male WT. However, in C57bL/6 mice, we described bizarre behavior that could be conspicuous at 6 months of age when confronting the anxiogenic environments such as the open-field test, mainly in females due to their increased anxious-like profiles as compared to males, or when these responses are found exacerbated by neuropathological conditions [10,44,46]. Here, the bizarre emergent behavior was vertical rearing, which resembles escape behavior in the behavioral despair test. More importantly, in the present work, this female pattern was emulated by homozygous PDK1 −/− and heterozygous PDK1 +/− male mice, to the extent that the male WT profile was dissonant with the one exhibited by all the other groups. We have shown that bizarre behaviors delay the exploratory activity in adults [44] and aged mice [46], and can be modulated by early-life interventions [44]. Therefore, the selective effects of sex and PDK/Akt signaling genotype-load on the vertical but not the horizontal activity, as shown by normal habituation curves, is noticeable and suggests that underlying mechanisms are mediated by anxiety but not by hyperactivity [47,48].

Performance of Mutants Can Resemble Normal under Naturalistic Anxiogenic Conditions
In the precedent work [36], we demonstrated that in the PDK1 homozygous mice, anxiety but not working memory was modulated by age with a reduction in its expression at 11-13 months of age. This would also explain that here, similarly to previous work [44], the anxiogenic environment of the open and illuminated field test was found to be the best to observe the elicitation of bizarre behaviors, as well the fine-tuning of genotype and sex modulation, but when the behavior of animals was assessed in the marble burying test, behaviors did not differ. Thus, in the current work, similar signatures were shown by the different groups and the 'moved or semi-buried marbles' was found the dominant behavioral readout at the end of the 30 min test. Compared to an anxiogenic open-field test, marble burying is a neuroethological paradigm eliciting spontaneous responses of vigorous and deep digging of beddings to bury the pieces (food pellets or small objects) the animal finds in its environment [49,50]. The interpretation of this test is in constant debate, since it is sensitive to anxiolytic but also antipsychotic drugs [45,51], and it is proposed for modeling compulsive-like characteristics of OCD or autism spectrum disorders [51,52]. Here, as in other research, its use was aimed to in vivo identification of biological impacts in mice [53,54], in our case of genetic load and sex. Furthermore, digging can also be understood as a measure of general activity rather than a measure of repetitive or anxiety-related behavior [51,[55][56][57] and conversely, the animal's general activity can be a confounding factor. Due to this controversy, the open-field or other anxiety tests that also monitor the general activity are a must for interpretations and discard confounding factors. In all cases, in the present work, the impact of sex and genetic load on specific vertical but not on locomotor activity may also agree with the similar signatures observed in this paradigm. This would also agree with reports on drug-induced dose-dependent reduction in marble-burying independently of its locomotor effects [58] or our most recent report in a model for neuropathological aging [47].

Conclusions
Of the two unconditioned tests used, the forced (open-field) but not naturalistic (marble arena) anxiogenic environmental conditions were sensitive to detect sex and geneticload differences at middle age in the PDK1 mutant mice. Thus, despite similar initial fear response (freezing indicating increased neophobia) and horizontal locomotion among the six groups of animals, females exhibited faster ethograms than males, with increased thigmotaxis with shorter latencies to reach the periphery and perform wall rearings, and increased wall and bizarre rearing. Genotype-load unveiled increased anxiety in males in elicitation of male ethograms and profiles resembling the performances characteristic of the female phenotype. While a heterozygous genotype-load was enough to elicit reduced somatic growth (bodyweight) in females, an homozygotic load of PDK1 was needed to exert this somatic effect in males. In summary, factor interactions indicated the complex interplay in the involvement of PI3K/Akt signaling pathway in the elicitation of different NVS's construct items and somatic growth and the relevance of genotype-load and sex in the fine-tuning of its intensity.