Slc6a20a Heterozygous and Homozygous Mutant Mice Display Differential Behavioral and Transcriptomic Changes

SLC6A20A is a proline and glycine transporter known to regulate glycine homeostasis and NMDA receptor (NMDAR) function in the brain. A previous study found increases in ambient glycine levels and NMDA receptor-mediated synaptic transmission in the brains of Slc6a20a-haploinsufficient mice, but it remained unknown whether Slc6a20a deficiency leads to disease-related behavioral deficits in mice. Here, we report that Slc6a20a heterozygous and homozygous mutant mice display differential behavioral phenotypes in locomotor, repetitive behavioral, and spatial and fear memory domains. In addition, these mice show differential transcriptomic changes in synapse, ribosome, mitochondria, autism, epilepsy, and neuron-related genes. These results suggest that heterozygous and homozygous Slc6a20a deletions in mice lead to differential changes in behaviors and transcriptomes.


INTRODUCTION
NMDA receptors (NMDARs) critically regulate the development and function of the nervous system (Paoletti et al., 2013;Zhu and Paoletti, 2015;Hansen et al., 2018). Glycine homeostasis is a key regulator of NMDAR function in the brain (Johnson and Ascher, 1987;Paoletti et al., 2013;Zhu and Paoletti, 2015;Hansen et al., 2018). Glycine levels in the extracellular spaces of the brain are thought to be regulated by two well-known glycine transporters, GlyT1 and GlyT2, which are encoded by Slc6a9 and Slc6a5, respectively (Smith et al., 1992;Liu et al., 1993).
SLC6A20A, which was originally reported to transport mainly proline (Smith et al., 1995;Nash et al., 1998;Kiss et al., 2002;Kowalczuk et al., 2005;Takanaga et al., 2005;Broer and Gether, 2012), was recently shown to also transport glycine and regulate glycine/proline homeostasis and NMDAR function in the mouse brain (Bae et al., 2021). Thus, SLC6A20A could be a novel target for inhibiting glycine uptake in the brain and thereby increasing ambient glycine levels and NMDAR function; this could be relevant for the treatment of NMDARrelated brain disorders (Bae et al., 2021), similar to the targeting of GlyT1 and GlyT2 for the treatment of schizophrenia, alcohol dependence, and pain (Tsai et al., 2004;Lane et al., 2006;Javitt, 2012;Harvey and Yee, 2013). However, it remains unclear whether deletion of Slc6a20a in mice leads to any disease-related behavioral deficits through specific mechanistic deviations.
In the present study, we show that Slc6a20a heterozygous and homozygous mice (Slc6a20a +/− and Slc6a20a −/− mice, respectively) display differential behavioral deficits in the locomotor, repetitive behavioral, and memory domains as well as transcriptomic changes in genes associated with synapses, mitochondria, ribosomes, autism spectrum disorders (ASD), epilepsy, and neurons.

Mice
Mice lacking exon 3 of the Slc6a20a gene were previously described (Bae et al., 2021). All mice were maintained under a 12-hr light/dark cycle (light phase: between 1:00 a.m. and 1:00 p.m.), given food and water ad libitum, and weaned at postnatal day 21. Female heterozygous mice and male heterozygous mice were crossed in order to obtain wildtype, heterozygous, and homozygous progenies. All mice were maintained in the mouse facility of Korea Advanced Institute of Science and Technology (KAIST). The experimental procedures were approved by the Committee of Animal Research at KAIST (KA2020-89).

Behavioral Tests
Adult male mice aged 3-6 months were used for all behavioral tests. All behavioral experiments were performed during the dark phase of the light/dark cycle (1:00 p.m. to 1:00 a.m.) in which mice are usually active. The brightness conditions in behavioral experiments described below refer to the local brightness around the center region of the apparatus rather than that in the behavioral room. Mice were habituated to an empty, dark experimental room for 30 min before the start of each behavioral test except for the Morris water maze test. EthoVision XT (Noldus) was used to analyze behavioral results unless otherwise noted.

Open-Field Test
This experiment was performed in order to analyze the locomotor activity and anxiety-like behavior in mice. A mouse was placed into the center of a white box (40 cm x 40 cm x 40 cm; ∼100 lux in the center). Mouse movements were then recorded for an hour, and the distance moved and time spent in the center region of the apparatus were analyzed.

Elevated Plus-Maze Test
This experiment was performed to investigate heightinduced anxiety-like behavior in mice. A heightened, cross-shaped device with two arms closed and two arms open was used for the experiment. A mouse was placed in the center region of the apparatus (∼200 lux) and allowed to freely move around the environment for 10 minutes. Time spent in closed arms, open arms, and center was analyzed.

Light-Dark Box Test
This experiment was performed to measure light-induced anxiety-like behavior in mice. The apparatus contained two different boxes/chambers, with one with the roof (dark box), and another without the roof (light box; ∼250 lux). A mouse was placed in the light box at the beginning and allowed to freely move around the environment.

Three-Chamber Test
This test was performed to measure social approach and social novelty recognition (Crawley, 2004;Nadler et al., 2004). A mouse was placed in the center chamber of the three-chambered apparatus (59 cm x 39.5 cm x 21.5 cm; ∼100 lux), and its activity was recorded for 10 min. Then, a stranger mouse (S1) was positioned in the cage placed at the corner of a side chamber, and an object (O) was placed in the cage at the corner of the other side chamber. 129S1 mice from the Jackson Laboratory were used as strangers. After 10-min recording of mouse activity, the object was replaced with another stranger (S2), followed by 10-min recording.

Morris Water Maze Test
This test was performed to measure spatial learning and memory in mice (Vorhees and Williams, 2006). A round pool was filled with white paint-added water. Water temperature was maintained at ∼20 • C. The pool was divided into four quadrants, and a platform was positioned in one of the quadrants. For the first six days, mice were taught to find an invisible platform. On the seventh day, the probe test was performed: a platform was removed from the pool. A mouse was then placed in the center of the pool and allowed to freely swim around for a minute. Time spent in quadrants and number of crossings across the platform were measured. After the probe test, a platform was placed in the opposite quadrant. For additional three days, mice were trained to locate the new position of the platform (reversal phase). On the eleventh day, another probe test (reversal probe test) was performed.

Contextual Fear Conditioning Test
This test was performed to measure fear learning and memory in mice. On day 0, a mouse was placed in the chamber with an electrocuting platform and habituated for 5 min. On day 1, foot shocks (0.8 mA) were provided for 2 s at 2, 3, and 4 min after the start of recording. Activity of a mouse was recorded for 5 min. On days 2, 3, and 8, a mouse was placed into the same chamber without any shock, and its activity was recorded for 5 min. Percent of freezing time was calculated.

LABORAS (Laboratory Animal Behavior Observation
Registration and Analysis System) experiments were performed for a long-term (4-day) monitoring of various mouse behaviors such as locomotor activity, repetitive behaviors (climbing, grooming, and rearing), drinking, and eating (Quinn et al., 2003(Quinn et al., , 2006. Total investigation time was 96 h with 12-h light/dark cycle, during which food and water were provided.

RNA-Seq Analysis
Four mice at ∼P120 were used for each group (wild type, heterozygous, and homozygous). The extracted mouse brains were preserved in RNAlater solution (Ambion) and stored at −20 • C. Poly-T oligo-attached magnetic beads were utilized to purify poly-A mRNAs. RNA concentrations were quantified using Quant-IT RiboGreen (Invitrogen, R11490), and RNA integrity was determined using TapeStation RNA screen tape (Agilent Technologies), after which only high-quality RNAs (RIN > 7.0) were selected for cDNA library construction using Illumina TruSeq mRNA Sample Prep kit (Illumina). Indexed libraries were submitted to an Illumina NovaSeq (Illumina), and paired-end (2 × 100 bp) sequencing was performed by Macrogen Inc.
Transcript abundance was estimated with Salmon (v 1.1.0) (Patro et al., 2017) in Quasi-mapping-based mode onto the Mus musculus genome (GRCm38) with GC bias correction (−gcBias). The acquired abundance data was imported to R (v.3.5.3) with tximport (Soneson et al., 2015) package and differential gene expression analysis was performed using R/Bioconductor DEseq2 (v1.30.1) (Love et al., 2014). Principal component analysis (PCA) was performed for the regularized log transform (rlog) of the normalized counts using plotPCA (with default parameter) tools implemented in DEseq2. Normalized read counts were computed by dividing the raw read counts by size factors and fitted to a negative binomial distribution. The p-values were adjusted for multiple testing with the Benjamini-Hochberg correction. Genes with an adjusted p-value of less than 0.05 were considered as differentially expressed. Volcano plots were generated using R ggplot2 (v.3.1.1) package. The Gene Ontology (GO) enrichment analyses were performed using DAVID software (version 6.8) (Huang da et al., 2009). Mouse gene names were converted to human homologs using the Mouse Genome Informatics (MGI) database. Gene Set Enrichment Analysis (GSEA) (Subramanian et al., 2005(Subramanian et al., , 2007 was performed to determine whether a priori-defined gene sets would show statistically significant differences in expression between WT and Slc6a20a-mutant mice. Enrichment Analysis was performed using GSEAPreranked R (gsea-3.0.jar) module on gene set collections downloaded from Molecular Signature Database (MSigDB) v 7.0. GSEAPreranked was applied using the list of all genes expressed, ranked by the fold change and multiplied by the inverse of the p-value with recommended default settings (1,000 permutations and a classic scoring scheme). The false discovery rate (FDR) was estimated to control the false positive finding of a given normalized enrichment score (NES) by comparing the tails of the observed and null distributions derived from 1,000 gene set permutations. The gene sets with an FDR of less than 0.05 were considered as significantly enriched. Integration and visualization of the GSEA results were performed using the EnrichmentMap Cytoscape App (version 3.8.1) (Merico et al., 2010;Isserlin et al., 2014).

Statistical Analysis
GraphPad Prism (version 9.2.0; GraphPad Software) were used to perform the statistical analyses. Outliers were retained. Statistical details are presented in Supplementary Table 1.

Slc6a20a Deficiency in Mice Induces Moderate Hyperactivity Without Affecting Anxiety-Like Behavior
Because glycine levels and NMDAR functions are elevated in Slc6a20a-mutant mice (Bae et al., 2021), and NMDARs critically regulate brain development and function (Paoletti et al., 2013), we subjected Slc6a20a heterozygous and homozygous mutant mice (Slc6a20a +/− and Slc6a20a −/− mice, respectively) to a battery of behavioral tests.
As the first step, we used immunoblot analysis to determine the temporal pattern of Slc6a20a protein expression during brain development. The immunoblot pattern from whole-brain lysates indicated that protein expression was strong at early developmental stages, including late embryonic and neonatal stages, and thereafter decreased to adult levels over the first few weeks of postnatal life ( Figure 1A).
In the open-field test, Slc6a20a +/− and Slc6a20a −/− mice (3-6 months) showed locomotor activities comparable to those of wild-type (WT) mice ( Figure 1B), suggestive of normal locomotor activity in a novel environment. In the Laboras test, where mouse movements were monitored for 4 consecutive days (Quinn et al., 2003(Quinn et al., , 2006, Slc6a20a +/− mice showed normal locomotor activity comparable to that of WT mice ( Figure 1C). In contrast, Slc6a20a −/− mice showed moderately increased locomotor activity during the total period and the light-on period, but not during the light-off period ( Figure 1C), suggesting that homozygous but not heterozygous Slc6a20a deletion in mice leads to hyperactivity in a familiar environment. The increased activity during the light-on period may also suggest that sleep was disturbed in Slc6a20a −/− mice, as reported previously in mice lacking the receptor tyrosine phosphatase PTPRS and showing disturbed sleep behaviors and rhythms (Park et al., 2020).
Slc6a20a +/− and Slc6a20a −/− mice spent normal amounts of time in the center region of the open-field arena ( Figure 1B), suggesting that anxiety-like behavior was not altered. Similarly, Slc6a20a +/− and Slc6a20a −/− mice showed normal levels of anxiety-like behaviors in the elevated plus-maze and light-dark tests (Figures 1D,E).
These results collectively suggest that homozygous but not heterozygous Slc6a20a deletion in mice induces moderate hyperactivity in a familiar environment, without altering anxietylike behavior.
FIGURE 1 | Slc6a20a deficiency in mice induces moderate hyperactivity without affecting anxiety-like behavior. (A) Temporal expression pattern of SLC6A20A proteins in mouse brains at the indicated embryonic (E) and postnatal (P) days. Whole-brain lysates were immunoblotting with SLC6A20 and control (GAPDH) antibodies. SLC6A20 signals were normalized to GAPDH signals for quantification. Note that the utilized pan-SLC6A20 antibody could recognize both SLC6A20A and SLC6A20B proteins in the brain; the expression level of SLC6A20A seems to be about three times greater based on the decrease in the pan-SLC6A20 signal of Slc6a20a homozygous mutant mouse brain (Bae et al., 2021)  , Student's t-test). Statistical significance and p values in the graphs; *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant.

Slc6a20a Deficiency in Mice Moderately Enhances Repetitive Behavior Without Affecting Social Behavior
We next subjected Slc6a20a-mutant mice to behavioral tests in the social and repetitive behavioral domains. In the threechamber social-interaction test, Slc6a20a +/− and Slc6a20a −/− mice displayed normal levels of social approach, as shown by time spent exploring social and non-social targets and the social preference index (Figure 2A). In addition, these mice displayed normal levels of social novelty recognition, as shown by time spent exploring novel and familiar social targets (Figure 2A).
In the Laboras test, Slc6a20a −/− but not Slc6a20a +/− mice showed increased repetitive climbing ( Figure 2B), a form of repetitive behavior characterized by overhanging from the wire FIGURE 2 | Slc6a20a deficiency in mice moderately enhances repetitive behavior without affecting social behavior. (A) Normal levels of social approach and social novelty recognition are seen in Slc6a20a +/− and Slc6a20a −/− mice (3-6 months) in the three-chamber test, as shown by the time spent exploring/sniffing a social stranger (S1) vs. an object (O) for social approach or a novel stranger (S2) vs. a familiar stranger (S1) for social novelty recognition, and also by the social preference index derived from sniffing time for S1 -O/sniffing time for S1 + O (or sniffing time for S2 -S1/sniffing time for S2 + S1 . Statistical significance and p values in the graphs; *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant. cage lids (Protais et al., 1976;Riffee et al., 1979;Wilcox et al., 1979;Cabib and Puglisi-Allegra, 1985). However, Slc6a20a +/− and Slc6a20a −/− mice did not show any alteration in other repetitive behaviors, such as self-grooming and rearing ( Figure 2B). Other behaviors, such as drinking and eating, were also normal in Slc6a20a −/− and Slc6a20a +/− mice (Supplementary Figure 1).
These results suggest that Slc6a20a deficiency in mice induces a moderate increase in repetitive behavior without affecting social behaviors, with repetitive climbing but not other repetitive behaviors increased in Slc6a20a −/− but not Slc6a20a +/− mice.

Slc6a20a Deficiency in Mice Moderately Enhances Spatial and Fear Learning and Memory
Because NMDARs critically regulate various forms of learning and memory (Collingridge, 1987;Bliss et al., 2014), we next subjected Slc6a20a +/− and Slc6a20a −/− mice to spatial and fear learning and memory paradigms.
In the Morris water maze test, Slc6a20a +/− and Slc6a20a −/− mice showed normal levels of spatial memory acquisition and retrieval during the initial learning and probe phases of the test, respectively, compared with WT mice (Figure 3A). In the reversal test, Slc6a20a +/− but not Slc6a20a −/− mice showed enhancements during the acquisition but not probe phase, compared with WT mice (Figure 3A).
In the contextual fear memory conditioning test, Slc6a20a +/− mice showed normal levels of fear memory acquisition on day 1 compared with WT mice (Figure 3B). In memory retrieval tests consecutively performed on day 2 (for 24-h retrieval), day 3 (for 48-h retrieval), and day 8 (7-day retrieval), Slc6a20a +/− mice showed largely normal fear memory retrieval.
Slc6a20a −/− mice showed normal levels of fear memory acquisition on day 1 (Figure 3B), although there was an increasing tendency. In the retrieval tests, Slc6a20a −/− mice showed increased retrieval on day 3 (48-h retrieval); the levels of retrieval on day 2 (24-h retrieval) and day 8 (7-day retrieval) were normal but showed increasing tendencies. These results collectively suggest that Slc6a20a deficiency in mice induces moderate increases in spatial learning and memory in the Morris water maze test and fear learning and memory in contextual fear conditioning test.
These results collectively suggest that both Slc6a20a +/− and Slc6a20a −/− mice show upregulation of genes that are associated with synaptic and neuronal functions and downregulation of genes associated with ribosomal and mitochondrial functions. Similar results were obtained from our gene set enrichment analysis (GSEA; see below).
Biological Functions Altered in the Transcriptomes of Slc6a20a +/− and Slc6a20a −/− Mice Revealed by GSEA Since our DEG analysis yielded small numbers of DEGs, we performed gene set enrichment analysis (GSEA), which can identify altered biological functions using a large number of small but coordinated transcriptomic changes in a less biased manner than analysis of a small number of large transcriptomic changes (Subramanian et al., 2005(Subramanian et al., , 2007. The transcripts derived from WT and Slc6a20a +/− mice (HT/WT transcripts; ∼P120 whole brain) were positively enriched for gene sets associated with neuronal synapses, as shown by the top five enriched gene sets ( Figure 5A and Supplementary Table 4). Clustering of the positively enriched gene sets using CytoScape Enrichment App (Merico et al., 2010;Isserlin et al., 2014) further confirmed that there were positive enrichments for functions associated with neuronal synapses, such as synaptic specialization, presynaptic active zone, ion channels, dendrites, and axons ( Figure 5B).
These results were obtained using the gene sets of the cellular component domain in the C5 database; we also obtained similar results using the gene sets of the biological process and molecular function domains of the C5 database ( Supplementary  Figures 2A,B, 3A,B and Supplementary Table 4). In addition, the use of gene sets in the KEGG domain indicated positive enrichments for synapse-related gene sets including longterm potentiation and gap junction, known to cooperate with excitatory synapses (Lee et al., 2021), as well as negative enrichments for ribosome/mitochondria-related gene sets (Supplementary Figures 4A,B and Supplementary Table 4).
The HT/WT transcripts were negatively enriched for gene sets associated with ribosomes and mitochondria ( Figure 5A and Supplementary Table 4). In addition, CytoScape Enrichment App analysis revealed similar negative enrichments for ribosome/mitochondria-related functions, such as ribosomal subunits and respiratory chain complex ( Figure 5B).
The transcripts derived from WT and Slc6a20a −/− mice (HM/WT transcripts) were positively enriched for gene sets associated with neuronal synapses, as supported by the top five gene sets and CytoScape Enrichment App clustering (Figures 5C,D and Supplementary Table 4). In addition, the HM/WT transcripts were negatively enriched for gene sets associated with ribosomes and mitochondria, as supported by the top five gene sets and CytoScape Enrichment App clustering (Figures 5C,D and  Supplementary Table 4). Similar results were obtained using the gene sets of the biological process and molecular function domains (Supplementary Figures 2C,D, 3C  These results collectively suggest that heterozygous and homozygous deletion of Slc6a20a in mice leads to similar increases in synapse-associated genes and similar decreases in ribosome-and mitochondria-related genes.
Using these gene sets, we performed GSEA using the HT/WT and HM/WT transcripts from Slc6a20a-mutant mice. The HT/WT and HM/WT transcripts were positively and similarly enriched for ASD-related gene sets that are downregulated in ASD, such as DEG_Down_Voineagu and Co-Exp_Down_M12_Voineagu ( Figure 6A and Supplementary Table 4), suggesting that both HT/WT and HM/WT transcripts exhibit patterns opposite those observed in ASD (termed anti-ASD hereafter).
The HT/WT and HM/WT transcripts were also positively and similarly enriched for all of the tested ASD-risk gene sets, including SFARI genes, FMRP targets (most strongly enriched), De Novo Missense, De Novo Variants, and AutismKB ( Figure 6A and Supplementary Table 4), and thus again exhibited an anti-ASD pattern.
When tested against gene sets associated with various brain disorders (DisGeNet 2 (Pinero et al., 2017(Pinero et al., , 2020, the HT/WT and HM/WT transcripts were positively and similarly enriched for ASD and epilepsy-related gene sets, and the HM/WT transcripts were positively and strongly enriched for substance use-related gene sets relative to the HT/WT transcripts (Figure 6B and Supplementary Table 4).
The HT/WT and HM/WT transcripts were positively enriched for gene sets associated with glutamate and GABA neurons ( Figure 6C and Supplementary Table 4), suggestive of anti-ASD transcriptomic patterns. However, the HT/WT and HM/WT transcripts were negatively and moderately enriched for gene sets associated with oligodendrocytes ( Figure 6C), suggestive of "ASD-like" or "pro-ASD" transcriptomic patterns. Intriguingly, the HT/WT and HM/WT transcripts displayed weak enrichments for astrocyte and microglia-related gene sets relative to those for glutamate/GABA neurons; thus we observed both positive and negative enrichments for microglia ( Figure 6C).
These results collectively suggest that heterozygous and homozygous Slc6a20a deletions in mice lead to largely anti-ASD transcriptomic enrichment patterns, as supported by the enrichment patterns for ASD-related/risk and cell-type-specific gene sets. In addition, the cell-type-specific transcriptomic changes induced by Slc6a20a deletion are stronger in neurons and oligodendrocytes relative to glial cells.

DISCUSSION
In the present study, we analyzed the behavioral and transcriptomic phenotypes of Slc6a20a +/− and Slc6a20a −/− mice. Our results revealed that these mice display differential hyperactivity, repetitive climbing, and moderately enhanced spatial and fear memory, as well as upregulation of synapse-related genes and downregulation of ribosomeand mitochondria-related genes. ASD-related transcriptomic changes are also observed, such as upregulation of ASD-related genes, including FMRP targets, and stronger upregulation of neuron-related genes compared to glia-related genes.
The behavioral phenotypes seem to be stronger in Slc6a20a −/− mice than in Slc6a20a +/− mice in some assays. For instance, Slc6a20a −/− mice are hyperactive in a familiar environment (Laboras results) and show increased self-grooming in Laboras cages and abnormally increased 48-hr fear memory, whereas Slc6a20a +/− mice do not exhibit such changes. However, spatial learning and memory is increased in Slc6a20a +/− but not in Slc6a20a −/− mice during the reversal but not the initial phase of the Morris water maze test. These results collectively suggest that heterozygous and homozygous Slc6a20a deletions in mice lead to differential behavioral phenotypes.
The mechanisms underlying these behavioral deficits are unclear. However, the RNA-seq results obtained from Slc6a20a +/− and Slc6a20a −/− mice indicate that synapserelated genes are upregulated. We note that Slc6a20a +/− mice have been shown to display increased NMDAR function at juvenile stages (Bae et al., 2021). In addition, the increased Slc6a20a expression in Pten-mutant mice has been causally associated with decreased ambient glycine levels and NMDAR function, and increased repetitive self-grooming (Bae et al., 2021). Moreover, neuroligin-3, which is upregulated in both Slc6a20a +/− and Slc6a20a −/− mice, has been associated with the regulation of NMDAR functions (Etherton et al., 2011;Polepalli et al., 2017;Zhang et al., 2017). It is thus tempting to speculate that altered NMDAR function in Slc6a20a +/− and Slc6a20a −/− mice might be associated with the behavioral deficits observed in these animals.
It should be noted that Slc6a20a expression at the protein level is much higher at late embryonic and early postnatal stages than at adult stages, while the behavioral experiments were mainly performed using mutant mice at ages around 3-6 months. Therefore, it is less likely that adult-stage deficiency of Slc6a20a directly causes the behavioral deficits, and the mechanistic deviations occurred at early developmental stages may have long-lasting impacts. Details on such mechanisms remain to be determined. However, given that Slc6a20a deficiency leads to NMDAR hyperfunction at juvenile stages (Bae et al., 2021), as mentioned above, and NMDAR function has profound impacts on the brain development and function across all developmental stages (Paoletti et al., 2013;Zhu and Paoletti, 2015;Hansen et al., 2018), it is probable that the NMDAR function impaired during early development have long-lasting effects on neuronal and synapse/circuit mechanisms that are associated with the observed behavioral deficits.
A notable transcriptomic change associated with the increased synaptic gene expression seen in Slc6a20a +/− and Slc6a20a −/− mice is the decreased expression levels of ribosomal and mitochondrial genes. This leads us to question whether ribosomal and mitochondrial gene downregulation are causally associated with the synaptic gene upregulation and behavioral deficits in the mutant mice. It has been shown that synaptic proteins are reciprocally related to protein synthesis in ASD (Santini and Klann, 2014). In addition, mitochondrial dysfunction has been linked to synaptic deficits (Li et al., 2004;Vos et al., 2010;Sheng and Cai, 2012; and ASD (Hollis et al., 2017;Frye, 2020;Rojas-Charry et al., 2021), and ribosomal dysfunction has been associated with ASD and epilepsy (Lombardo, 2021). These results suggest that ribosomal and mitochondrial gene downregulation observed in our mutant mice may contribute to their synaptic and behavioral deficits.
GSEA using brain disorder-related gene sets shows that the Slc6a20a +/− and Slc6a20a −/− transcriptomes are associated with ASD and epilepsy, and that the Slc6a20a −/− transcriptome is more strongly associated with substance use-related disorders than the Slc6a20a +/− transcriptome. Among the ASD-related gene sets, the Slc6a20a +/− and Slc6a20a −/− transcriptomes are more strongly enriched for FMRP targets associated with the fragile X syndrome. These results suggest that Slc6a20a deletion in mice leads to transcriptomic changes associated with multiple brain disorders. In addition, given that many FMRP targets have been related to synaptic functions (Bagni and Zukin, 2019), these results further support the hypothesis that synaptic deficits may underlie the behavioral deficits in these mutant mice.
GSEA using cell-type-specific gene sets indicates that the Slc6a20a +/− and Slc6a20a −/− transcriptomes are more strongly enriched for neuron (glutamate and GABA)related gene sets relative to glia (astrocyte/microglia)-related gene sets. Slc6a20a has been shown to be expressed more strongly in glial cells (astrocytes and microglia) than in neurons (glutamate and GABA) (Bae et al., 2021). Our results suggest that the transcriptomic changes observed in synapse/ribosome/mitochondria-related genes in the mutant brain likely represent changes occurring in neurons rather than cell-autonomous changes occurring in glial cells (astrocytes and microglia).
Lastly, it should be pointed out that the differences between Slc6a20a +/− and Slc6a20a −/− mice were greater in behavioral phenotypes than in transcriptomic phenotypes. It is possible that the subtle differences between Slc6a20a +/− and Slc6a20a −/− mice in the expression levels of neuron/gliarelated genes (i.e., different cortical layers, GABA subtypes, and glial cell types) may still contribute to the differential behavioral changes. Alternatively, the transcriptomic changes may reflect compensatory changes initiated in an effort to rescue the gene deletion effects, rather than those mediating the pathophysiological changes. Further validation of the mechanistic/functional changes suggested by the transcriptomic changes should be tested at the protein, synaptic, and functional levels.
In summary, we herein show that heterozygous and homozygous Slc6a20a deletions in mice lead to differential behavioral deficits in locomotor, repetitive behavioral, and spatial and fear memory domains and transcriptomic changes in genes associated with synapses, ribosomes, mitochondria, ASD, epilepsy, and neurons.

DATA AVAILABILITY STATEMENT
The RNA-Seq data presented in the study are deposited in the Gene Expression Ombinus (GEO) repository at the National Center for Biotechnology Information (NCBI), accession number GSE193387.

ETHICS STATEMENT
The animal study was reviewed and approved by Committee of Animal Research at KAIST (KA2020-89).

AUTHOR CONTRIBUTIONS
JK, JR, and MB performed behavioral experiments. SK performed immunoblot analysis. HK performed RNA-Seq analyses. MB, HK, and EK wrote the manuscript. All authors contributed to the article and approved the submitted version.

FUNDING
This work was supported by the Korea Institute of Science and Technology Information (K-19-L02-C07-S01 to HK) and the Institute for Basic Science (IBS-R002-D1 to EK).