Neuron-specific ablation of eIF5A or deoxyhypusine synthase leads to impairments in growth, viability, neurodevelopment, and cognitive functions in mice

Eukaryotic initiation factor 5A (eIF5A)†, ‡ is an essential protein that requires a unique amino acid, hypusine, for its activity. Hypusine is formed exclusively in eIF5A post-translationally via two enzymes, deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase. Each of the genes encoding these proteins, Eif5a, Dhps, and Dohh, is required for mouse embryonic development. Variants in EIF5A or DHPS were recently identified as the genetic basis underlying certain rare neurodevelopmental disorders in humans. To investigate the roles of eIF5A and DHPS in brain development, we generated four conditional KO mouse strains using the Emx1-Cre or Camk2a-Cre strains and examined the effects of temporal- and region-specific deletion of Eif5a or Dhps. The conditional deletion of Dhps or Eif5a by Emx1 promotor–driven Cre expression (E9.5, in the cortex and hippocampus) led to gross defects in forebrain development, reduced growth, and premature death. On the other hand, the conditional deletion of Dhps or Eif5a by Camk2a promoter–driven Cre expression (postnatal, mainly in the CA1 region of the hippocampus) did not lead to global developmental defects; rather, these KO animals exhibited severe impairment in spatial learning, contextual learning, and memory when subjected to the Morris water maze and a contextual learning test. In both models, the Dhps-KO mice displayed more severe impairment than their Eif5a-KO counterparts. The observed defects in the brain, global development, or cognitive functions most likely result from translation errors due to a deficiency in active, hypusinated eIF5A. Our study underscores the important roles of eIF5A and DHPS in neurodevelopment.

Polyamines (putrescine, spermidine, and spermine) are essential for eukaryotic cell growth and regulate a vast array of cellular activities (9)(10)(11). Polyamine homeostasis is tightly regulated by intricate mechanisms at multiple levels including biosynthesis, catabolism, and transport. The majority of cellular polyamines are bound to RNA, and the most important function of polyamines appears to be the regulation of translation as polycations (10,12) and also as a component of hypusine in eIF5A. As hypusine is vital for eIF5A activity and cell proliferation, hypusine synthesis defines a critical function of polyamines in eukaryotic cell growth (13,14).
In discrepancy to its nomenclature, eIF5A facilitates translation elongation rather than translation initiation (15)(16)(17). In yeast, eIF5A promotes translation elongation broadly at ribosome stall sites including sequences encoding polyproline stretches, and it also enhances translation termination (15,18). eIF5A binds to the 80S ribosome between the peptidyl-tRNA site and the exit tRNA site (19). The hypusine side chain of eIF5A stabilizes the binding of the peptidyl tRNA to the 80S ribosome and facilitates peptide bond synthesis. Two or more eIF5A isoform genes have been identified in many eukaryotic organisms from fungi to humans. In the human and mouse, eIF5A2 shares 84% and 82% amino acid sequence identity with eIF5A1 (usually termed eIF5A), respectively. Both isoforms * For correspondence: Myung Hee Park, mhpark@nih.gov. † The nomenclature has changed from IF-M2Ba to eIF-4D to the current eIF5A ‡ As eIF5A mostly exists as the hypusinated form in cells or tissues, eIF5A usually denotes natural eIF5A containing hypusine. eIF5A may also designate all forms of eIF5A with different modification status.
effectively undergo hypusine modification in cells (20). § However, only eIF5A1 is constitutively expressed in all mammalian cells and tissues, whereas the eIF5A2 isoform mRNA expression appears to be tissue specific in the brain and testis (21). The eIF5A2 protein is normally undetectable in most mammalian tissues and cells, but increased expression of this isoform or eIF5A has been associated with various human cancers (22)(23)(24). The Eif5a2 homozygous KO mouse develops and grows normally, suggesting that eIF5A2 is dispensable for mouse development (25). DHPS is known to be totally specific for eIF5A (eIF5A1 and eIF5A2 isoforms); no other cellular protein is modified by DHPS. The exclusive specificity is based on the requirement for a macromolecular interaction between DHPS and the nearly intact N domain of eIF5A. A potential role of eIF5A and DHPS in neuronal growth and survival was first suggested in studies that used the neuronal cell line PC12 and rat primary hippocampal cultures in vitro (26). In these studies, a reduction of hypusinated eIF5A by using a DHPS inhibitor or DHPS RNAi attenuated neurite outgrowth and neuronal survival (26). Only recently, definitive genetic evidence for their importance in human neurodevelopment was reported (27)(28)(29). From whole-exome sequencing and genetic analysis, biallelic DHPS variants were identified as the cause of a rare autosomal recessive neurodevelopmental disorder (28). More recently, germ line, de novo, heterozygous EIF5A variants were also reported to be associated with a neurodevelopmental disorder (27). The patients carrying biallelic DHPS variants, or heterozygous EIF5A variants, share common phenotypes including intellectual disability and developmental delay. In addition, among the five DHPS variant patients, four have facial dysmorphism, one has microcephaly, and four have clinical seizures. Of the seven EIF5A variant patients, all display facial dysmorphism and five of them with microcephaly. Thus, a decrease in the biologically active, hypusinated form of eIF5A appears to interfere with proper neurodevelopment in humans.
To further investigate the roles of eIF5A and DHPS in brain development, we have generated four mouse strains in which either Eif5a or Dhps is deleted in the brain in a temporally and spatially specific manner using the Emx1-Cre or the Camk2a-Cre line. Phenotype analyses revealed severe morphological defects in the brain, growth retardation, and reduced viability in mice with Emx1-Cre-mediated deletion of Eif5a or Dhps, and impaired cognitive functions in mice with Camk2a-Cre-mediated deletion of Eif5a or Dhps.
The effects of temporal-and region-specific KO of Eif5a or Dhps in the brain on growth and survival of mice In the Emx1-Cre-driven KO strains, the Eif5a or Dhps gene is downregulated in the neurons of the developing rostral brain including the cerebral cortex, and hippocampus, beginning at E9.5 and continuing throughout postnatal life. On the other hand, in the Camk2a-Cre-driven CKO strains, the expression of the target gene is abolished postnatally (beginning at P15-P21 and continuing through adulthood) in the Camk2aexpressing neurons in the CA1 regions of the hippocampus (31). Differential phenotypes were observed in all four CKO strains. Both male and female groups of Eif5a Emx pups grew significantly slower than the control Eif5a fl/fl pups (Fig. 3, A  and B). There was little difference in the growth rates between the male and female groups of the Eif5a Emx mice, whereas in the control Eif5a fl/fl group, the males were consistently heavier than the female counterparts (Fig. 3, A and B). Moreover, survival was reduced in Eif5a Emx mice compared with the control mice (Fig. 3C). The average body weights of both the male and female Eif5a Emx mice were reduced compared with those of the control Eif5a fl/fl mice throughout the period examined (Fig. 3, A-D) Figure 1. The pathways of polyamine biosynthesis and hypusine modification in eIF5A. The abbreviations are as follows: DHPS, deoxyhypusine synthase; DOHH, deoxyhypusine hydroxylase; ODC, ornithine decarboxylase; SMS, spermine synthase; SRM, spermidine synthase. § In mammals, there are two eIF5A genes, encoding highly conserved isoforms, eIF5A1 and eIF5A2, and both undergo hypusine modification. As eIF5A1 is the isoform predominantly expressed, eIF5A commonly represents eIF5A1. eIF5A can also be used to represent both forms collectively.
At birth, the Dhps Emx mice appeared to be similar in size to the control mice (Dhps fl/+ , Emx1-cre, Dhps fl/fl , Dhps fl/+ ). However, the postnatal growth of Dhps Emx mice was significantly impaired (Fig. 4A) and nearly arrested by day 12, whereas the control mice continued to grow. All Dhps Emx pups died before 4 weeks after birth (Fig. 4B). On day 24, Dhps Emx mice were much smaller than Dhps fl/fl mice with the average whole-body weight less than 50% of the control mice (Fig. 4C). Unlike the deletion of Eif5a or Dhps in the Emx1-expressing neurons, deletion of either gene in the Camk2a-expressing neurons did not result in significant inhibition of growth, and no visible signs of developmental defects were observed in the first 3 months. However, both Eif5a Camk2a and Dhps Camk2a mice lost viability between 2 and 9 months of age (Fig. S1).
The effects of deletion of Dhps or Eif5a on brain development and morphology The deletion of Eif5a or Dhps also exerted variable impacts on brain development in the four CKO strains (Figs. 5 and 6). Gross brain images revealed quite similar morphological defects in the brains of Eif5a Emx and Dhps Emx mice (Fig. 5, A and C), although Dhps Emx mice displayed more serious defects in growth and survival than Eif5a Emx mice. The average brain weight of the Eif5a Emx mice at 4 months was less than that of controls (0.25 g versus 0.48 g, respectively). The same gross lesions shown in Figure 5A were observed in all Eif5a Emx brains examined (1-and 4-month-old mice). The abnormal brain morphology included the loss of the cerebral cortex, hippocampus, corpus callosum, internal capsule, and portions of the lateral ventricles, and the opening of the third ventricle to the meninges. However, we could not detect cellular  changes in the microscopic images of the remaining part of the Eif5a Emx brain at 4 months ( Fig. 5I versus Fig. 5J). The average weight of the Dhps Emx brains was less than half of the control brain (0.19 g versus 0.431 g) on day 24. Each of the four Dhps Emx brains examined showed the same gross abnormality (Fig. 5C), similar to that of the Eif5a Emx brain (Fig. 5A). In the Dhps Emx brain, the rostral portion of the cerebral cortex was missing or thinned. The deformity also included agenesis of the corpus callosum, hippocampus, internal capsule, and the distal portion of the cerebrum overlying the mid-brain. The lumen and the roof of the third ventricle were missing. Microscopic images of the remaining Dhps Emx brain cerebrum showed the neurons enlarged and vesiculated (black arrows, Fig. 5K), not found in the control brain ( Fig. 5L).
In contrast to the Eif5a Emx and Dhps Emx mice, Eif5a Camk2a and Dhps Camk2a mice appeared to grow normally and their gross brain images were indistinguishable from those of control mice (Fig. S2). However, microscopic examination revealed that Dhps Camk2a mice had neuronal necrosis of the cerebral cortex and hippocampus at 4 months (  Interestingly, the immunostaining of glial fibrillary acidic protein, a marker of astrocyte, known to increase at the site of neuronal damage, appeared increased in the brain tissue sections from the Eif5a Camk2a , Dhps Emx , and Dhps Camk2a mice at multiple postnatal ages (Fig. 7A). Furthermore, terminal deoxynucleotidyl transferase dUTP nick end labeling assays performed on brain tissue sections from the mutant mice and controls also showed the appearance of apoptotic cells (Fig. 7B).

Impaired cognitive functions in the Eif5a Camk2a and Dhps Camk2a mice
We first compared the mobility of the Eif5a Camk2a mice with that of the control mice by the open field test. The distance traveled in the whole arena, the mobile time, and the speed of the CKO mice were not reduced compared with the controls (Fig. S3), suggesting that their mobility was not impaired. Furthermore, all the Eif5a Camk2a and Dhps Camk2a mice displayed normal swimming ability 1 day before the Morris water maze (MWM) test. In the MWM test (Fig. 8), the mouse relies on visual cues to navigate to a submerged escape platform. Spatial learning was assessed by daily repeated trials for 6 days. The latencies to reach the hidden platform for the two controls, Eif5a fl/fl and Dhps fl/fl , were 37.02 and 38.61 s, respectively, on day 1 and were shortened to 9.52 and 9.93 s, respectively, by day 6 (Fig. 8, A and B). On the other hand, the latencies of Eif5a Camk2a and Dhps Camk2a mice on day 1 (42.71 and 53.66 s, respectively) were longer than those of the respective controls, suggesting a poor baseline performance. Furthermore, the improvements of Eif5a Camk2a and Dhps Camk2a mice from day 1 to day 6 (reduction of latency by 53% and 38.5%, respectively) were much less than those of the respective control mice (reduction of latency by 74.3% and 75.1%, respectively), suggesting impaired learning in both CKO mice, with DHPS Camk2a showing greater impairment than EIF5A Camk2a . The analyses of the swim distance to the hidden platform also provided a similar indication of learning disability of the two CKO strains (Fig. 8, C and D). The swim distances were similar for all four groups on day 1 but were significantly longer for the Eif5a Camk2a and Dhps Camk2a mice than their respective controls on consecutive days. The improvement indicated by a shortening of the swim distance from day 1 to day 6 was worse for the CKO groups than their controls, and Dhps Camk2a consistently underperformed Eif5a Camk2a mice. These results provide strong evidence that both Eif5a Camk2a and Dhps Camk2a mice are impaired in spatial learning and that the impairment is more severe in Dhps Camk2a than in Eif5a Camk2a mice (Fig. 8, A-D).
After the completion of the 6-day trials, reference memory was evaluated by a probe trial after the removal of the hidden platform. The percentage time occupancy in the target quadrant (North West [NW]) (Fig. 8, E and F) B B B B B B B B B B B B B B B B  Role of eIF5A hypusination in mouse neurodevelopment crossings into the small area that previously contained the removed platform were measured (Fig. 8, G and H) trial, the mice were placed in the pool at the boundary of the North East and South East quadrants, or east start point. The control mice quickly and more directly moved to the NW quadrant to search for the platform location, suggesting good memory of the location while the mutant mice spent more time searching in the North East and South East quadrants before moving to search in the NW location, as shown in the track plots (Fig. S4), consistent with poor memory of the platform location during the probe trial. The average numbers of crossings into platform area were lower in the CKO mice than in the controls (7.42 and 4.94, respectively, for Eif5a fl/fl and Eif5a Camk2a mice and 6.29 and 2.08, respectively, for Dhps fl/fl and Dhps Camk2a mice). Both the platform occupancy and the platform area entry data provide clear evidence for memory impairment in the CKO mice, Dhps Camk2a mice being more deficient than Eif5a Camk2a mice. Then, a contextual learning (cued fear conditioning) test was carried out as outlined in the top panel of Figure 9. The baseline freezing and novel context baseline freezing were low and no significant differences were observed among the four groups of mice. However, contextual freezing time was significantly reduced in Eif5a Camk2a mice (to 48% of the control Eif5a fl/fl value) and Dhps Camk2a (to 40% of Dhps fl/fl value) (Fig. 9, A and B). Auditory cued freezing was also reduced in Eif5a Camk2a mice (to 64% of the control) and in Dhps Camk2a mice (to 78% of the control), but not as much as the contextual freezing. Taken together, the data in Figures 8 and 9 clearly demonstrate the impairment in spatial learning, memory, and contextual learning in mice in which Eif5a or Dhps is deleted in the Camk2a-expressing neurons of the cortex and hippocampus.

Discussion
Recent genetic studies have provided evidence that certain variants in the EIF5A (27) or DHPS gene (28) are associated with rare neurodevelopmental disorders in humans. Furthermore, individuals with DOHH variants who display similar developmental delay and intellectual disability have also been identified (Ziegler A. et al., unpublished results), underscoring the importance of each step of the hypusine modification pathway and thereby the critical role of the hypusinated eIF5A in neurodevelopment in humans (29). These findings led us to generate the four CKO mouse models, with temporal-and region-specific deletion of either Eif5a or Dhps in the forebrain, and to assess the impact on development. Different phenotypes in brain development, growth, survival, and cognitive functions were observed in these CKO strains depending on the targeted gene and the Cre driver. Although the CKO strains do not harbor the same variants of the EIF5A and the DHPS genes as those of the affected human individuals, it is interesting to note that certain features of the human neurodevelopmental disorders, including intellectual disability, developmental delay, reduced growth, and shortened lifespan, are reflected in the phenotypes of these CKO mice.
The deletion of Eif5a or Dhps in the Emx1-expressing neurons from the mid-embryonic stage resulted in gross morphological abnormalities in the brain; the cerebral cortex was thinned or missing and the hippocampus, corpus callosum, and the internal capsule portions of the ventricles were also missing because of agenesis (Fig. 5, A and C). These results indicate that both Eif5a and Dhps are essential for the embryonic and postnatal development of the cortex and hippocampus. Although gross brain defects were similar in the Eif5a Emx and the Dhps Emx mice, the deleterious effects on growth and viability were more severe in Dhps Emx mice than in Eif5a Emx mice. Approximately 67% of Eif5a Emx mice survived longer than 3 months, whereas all Dhps Emx mice died within 4 weeks after birth. The behavioral tests were not performed on these groups of CKO mice because of their short life spans and premature and unpredictable death, especially of Dhps Emx mice.
Whole-body KO of Eif5a or Dhps causes an early embryonic lethality (between E3.5 and E6.5) (6). It is predicted that eIF5A and DHPS would be depleted in the cortex and hippocampus area within a few days after Emx1-Cre expression at E.9.5. Given this timeline for gene deletion and the resultant observed phenotype, the mechanisms underlying the loss of the cortex and hippocampus may involve the failure of neural stem cells to proliferate or to differentiate, apoptosis of differentiating cells, or a failure of differentiating cells to proliferate. Previous studies using mouse and zebrafish models of DHPS knockdown during the developmental and postnatal periods have demonstrated that loss of DHPS impacts mRNA translation, which in turn disrupts cellular differentiation, organ development, and/or cellular proliferation (7,8,32). Therefore, as a translation elongation factor affecting synthesis of a wide array of cellular proteins, depletion of hypusinated   Figure 9. Impaired contextual learning in the Eif5a Camk2a and Dhps Camk2a mice. (A, B), The contextual learning test was performed with the two CKO mice and their control mice, as described in Experimental procedures. Baseline freezing, contextual freezing, novel context freezing, and auditory cue freezing were measured. The values are indicated as closed circles (controls) and open circles (CKO), and each bar represents the mean ± SD. The numbers of mice in each group were Eif5a Camk2a (n = 11), Eif5a fl/fl (n = 14), Dhps Camk2a (n = 9), and Dhps fl/fl (n = 19). The error bars represent the SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 via Student's t test. CKO, conditional KO. eIF5A may affect multiple cellular processes in brain development.
The postnatal ablation of Eif5a or Dhps in the Camk2aexpressing neurons did not cause gross changes in the brain compared with those found in the Eif5a Emx and the Dhps Emx mice, suggesting that the development of the cortex and hippocampus was unaltered. Although no significant growth inhibition or visible defects were found in Eif5a Camk2a or Dhps Camk2a mice, they both died prematurely between 2 and 9 months. Furthermore, they displayed concrete evidence of impairment in cognitive functions (Figs. 8 and 9), Dhps Camk2a being more affected than Eif5a Camk2a mice. These cognitively impaired CKO mice hold potential utility in the future development of chemical or biological therapeutics for human neurodevelopmental disorders caused by variants of EIF5A, or DHPS.
Common phenotypes between Eif5a Emx and Dhps Emx mice and between Eif5a Camk2a and Dhps Camk2a mice strongly suggest that a common mechanism underlies the impairment in both CKO mice. However, it is hard to explain why the ablation of Dhps is more detrimental than that of Eif5a, as is evident in all the observed phenotypes. This is counterintuitive, as the hypusinated eIF5A is the direct player in translation elongation, whereas DHPS is a modifier of eIF5A activity. One possibility may be that the eIF5A2 isoform (modified to the hypusine form) can partially compensate for the loss of eIF5A in the targeted neurons of the mouse brain. However, we did not find clear evidence for accumulation of the eIF5A2 isoform protein in brain tissues of control or CKO mice (data not shown). The whole-body KO of eIF5A is embryonic lethal in mouse, suggesting that eIF5A2 is not induced during early embryonic development upon KO of eIF5A and that eIF5A2, in this scenario, may not compensate for the loss of eIF5A (6). It is also possible that there are differences in the efficiency of the Cre-mediated recombination at the two different target gene loci. Whereas knockdown of expression of eIF5A or DHPS has been carefully validated for both conditional mouse alleles using other cell-specific cre-mediated models (7,8) and the efficiency of the Emx1-cre and Camk2a-cre drivers has been published (30,31), our study did not perform a direct comparison of the recombination efficiencies of these alleles in our models. Another possibility is the interference of activities of hypusinated eIF5A by unhypusinated eIF5A precursors that accumulate upon depletion of spermidine or upon inhibition of DHPS. Although the unhypusinated eIF5A precursors were inactive and did not appear to interfere with the activity of hypusinated eIF5A in the in vitro assays of methionylpuromycin synthesis (33), their potential effects on translation need to be reevaluated in vivo. It is possible that the eIF5A precursors still associate with the 80S ribosome through interactions not involving the hypusine residue (19) and interfere with the action of hypusinated eIF5A in mammalian cells and tissues. In such a case, the potential interference by the eIF5A precursors that may have accumulated in Dhps Emx and Dhps Camk2a brains could explain their more deleterious phenotypes. In the case of human patients, a heterozygous de novo EIF5A variant with partial activities causes clinical phenotypes (27), suggesting that proper neuronal function in humans cannot tolerate even a partial loss (<50%) of eIF5A activity. The detrimental effects of heterozygous EIF5A variants may not be simply due to a reduction in active eIF5A but may also be compounded by the interference by the eIF5A variants. The molecular basis underlying the better survival and performance of the Eif5a CKO mice than the Dhps CKO mice warrants further investigation.
The implication of variants of EIF5A or DHPS in human neurodevelopmental disorders is not surprising in view of the fact that variants in a number of other factors in the translation machinery such as alanyl tRNA synthetase and eukaryotic translation elongation factors 2 (EF2) and 1a2 (EF1a2) have been associated with neurodevelopmental disorders (34). Errors during translation elongation can lead to production and accumulation of aberrant proteins that are toxic to neural cells. In human individuals with variants in EIF5A, or DHPS, major clinical symptoms were associated with neurodevelopment (27,28), suggesting that neuronal systems are most vulnerable to a deficiency in hypusinated eIF5A. Global proteomics analyses provided evidence that depletion of eIF5A in mammalian cells led to endoplasmic reticulum stress, unfolded protein response, and upregulation of chaperone expression (35). In addition to these general effects of eIF5A depletion, it is also possible that there are key regulatory factors of brain development that may be specifically dependent on eIF5A for translation. Future studies will be directed toward elucidation of molecular mechanisms underlying these neurodevelopmental disorders stemming from a reduction in bioactive, hypusinated eIF5A and the identification of downstream effectors of eIF5A.

Mouse maintenance and sample collection
All experimental procedures involving mice were approved by the National Institute of Dental and Craniofacial Research Animal Care Committee and were conducted in accordance with approved protocols. Pups were housed in an animal facility with a 14/10 h light/dark cycle in positive pressureventilated racks with filtered-top cages (Lab Products Inc). Animals were fed autoclavable rodent pellets, NIH-07 Mouse/ Rat Diet (Envigo, #7022) and UV-treated ultra-filtered water ad libitum throughout the experiments. To help the feeding of small mutant mice, hydrogel and soft foods were offered in petri dishes on the floor of the cages.
Mouse strains used for brain-specific KO of Eif5a or Dhps The conditional mouse strains used, Eif5a fl/fl and Dhps fl/fl (Dhpstm1.1Mirm/J, stock #034895, Jackson Laboratory), were previously reported (7,8). Neither the Eif5a fl/fl mice nor Dhps fl/fl mice showed phenotypic differences compared with their WT C57BL/6 littermates, and as a result, Eif5a fl/fl and Dhps fl/fl mice were used as controls for the respective CKO strains. The homozygous Emx1-IRES-Cre (B6.129S2-Emx1, Jackson Laboratory) (30) were viable, fertile, and normal in size and did not display any gross physical or behavioral abnormalities. Recombination occurs in approximately 88% of the neurons of the neocortex and hippocampus and in the glial cells of the pallium starting at E.9.5. The Camk2a transgenic strain, T29-1 (B6.Cg-Tg(Camk2a-Cre), Jackson Laboratory) used in the study displayed a normal phenotype and the Camk2a-Cre recombinase was expressed in the forebrain, predominantly in the CA1 pyramidal cell layer in the hippocampus postnatally 3 to 4 weeks after birth (31).

Genotyping of KO mice
Genomic DNA was isolated from tail biopsies using the QIAamp DNA Blood Mini Kit (QIAGEN) according to the manufacturer's instruction. For PCR analysis, mouse tail DNA was amplified using JumpStart Taq ReadyMix (Milli-poreSigma). The Dhps lox PCR and Eif5a lox PCR were carried out as described previously (7,8).

Histochemical analysis
The animals were euthanized with carbon dioxide. A necropsy was performed, and multiple tissues and organs were collected and placed in 10% formalin and fixed for 24 to 48 h. The tissues were then processed through a series of alcohols and xylenes and embedded in paraffin.
Serial sections (thickness of 10 μm) were prepared and stained with 0.1% H&E or used for immunohistochemistry at Histoserv Inc as follows: Slides were deparaffinized and hydrated through graded alcohols to distilled water, followed by antigen retrieval. They were then blocked with hydrogen peroxide and a blocking serum, and slides were washed in distilled water. Next, the slides were incubated with the primary antibody, a secondary antibody, and horseradish peroxidaseconjugated streptavidin. Finally, the slides were developed using 3,3 0 -diaminobenzidine and counterstained with hematoxylin. All of the incubations were carried out at room temperature (RT) and TBST was used as a washing buffer.

Terminal deoxynucleotidyl transferase dUTP nick end labeling assays
Formalin-fixed paraffin-embedded brain tissue sections mounted on glass slides were deparaffinized and hydrated through graded alcohols to distilled water, followed by proteinase digestion at 37 C. Tissue sections were then blocked with a blocking serum, rinsed, and transferred to a buffer solution. Next, the slides were incubated in the TdT/dUTP reaction mixture at 37 C. The tissue sections were again blocked with a blocking serum, rinsed, and detected with an anti-digoxigenin detection system. Finally, the slides were developed with Vector Red and counterstained with hematoxylin. Unless otherwise specified, incubations were performed at RT, and TBST was used as a washing buffer.

Open field test
To measure the general activities of mice, the open field test was performed as follows: mice were removed from their home cages and gently placed in a 16" × 16" × 16" Perspex arena viewing chamber and their movement was recorded for periods ranging from 5 min to 30 min, and then, the mice were returned to their standard home cages. The recordings were fed into a software program that analyzes the mouse behavior and movement.

MWM test
Learning plasticity and cognitive flexibility were tested by the MWM test (36) with minor modifications. Although the ages of tested mice varied from 2.5 to 5 months, a mutant and a matching control (with the same or close to the same age) were set up to be tested in pairs. The MWM test was carried out in a circular pool (4 ft. diameter, 30 in. high, San Diego Instruments) filled with water, which was made opaque with addition of non-toxic white paint (Crayola) and kept at 20 to 30 C. A small, square, clear plexiglass escape platform (hidden platform) was placed in the NW quadrant of the tank, 1 cm beneath the water surface. The swim distance, latency to find the platform, swim speed, path length to platform, and so forth were measured using behavioral tracking software (ANYmaze). The mice received four learning trials per day (trials lasted maximum of 60 s) on six consecutive days. After learning trials were completed, a probe trial was performed on the last day, in which the platform was removed. The number of crossings over the location in the pool previously occupied by the removed platform and the percentage of time spent in each of the four pool quadrants were measured for 90 s.

Contextual learning test
The test was conducted following guidelines of a published protocol (37) with modifications. While the animal was in the chamber and provided cues, the following data including the total freezing time, number of freezing episodes, duration of freezing episodes, and latency between stimuli and freezing were collected. Mice were placed in a sound-attenuating chamber, 17 cm × 17 cm × 25 cm (w, d, h) with a light and speaker. After baseline freezing was measured for 120 s, auditory tones (2 × 4 kHz 80 dB tone) were delivered into the chamber for 15 s, followed by a 2-s foot shock (0.85 mA) through the grid floor. After a break for 120 s, the tone-shock procedure was repeated, and the mice were returned to their cages. On day 2 (24th hour), the mice were re-exposed to the chamber used on day 1 and contextual freezing was recorded. Afterward (25th hour), the mice were placed in a completely new chamber and novel context freezing was measured for 120 s. Then, auditory cues were applied and auditory cue freezing was measured.

Statistics
All data are presented as the mean ± SEM and were analyzed using the software GraphPad Prism 5.0 (GraphPad Software) and OriginPro (OriginLab Corp). Student's t test was applied to determine the significance between two groups. ANOVA with the post hoc Tukey test was used to analyze the time in each chamber in the social behavior test and the time in quadrants in the MWM. Two-way ANOVA was applied to compare the probe trial data in the MWM and in the contextual learning test. Statistical significance was defined at p < 0.05 and presented as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Data availability
All the data described are contained within the article.
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