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. 2023 Dec 21;18(12):e0295855. doi: 10.1371/journal.pone.0295855

Social isolation-induced transcriptomic changes in mouse hippocampus impact the synapse and show convergence with human genetic risk for neurodevelopmental phenotypes

Aodán Laighneach 1, John P Kelly 2, Lieve Desbonnet 2, Laurena Holleran 1, Daniel M Kerr 2, Declan McKernan 2, Gary Donohoe 1, Derek W Morris 1,*
Editor: Alexandra Kavushansky3
PMCID: PMC10735045  PMID: 38127959

Abstract

Early life stress (ELS) can impact brain development and is a risk factor for neurodevelopmental disorders such as schizophrenia. Post-weaning social isolation (SI) is used to model ELS in animals, using isolation stress to disrupt a normal developmental trajectory. We aimed to investigate how SI affects the expression of genes in mouse hippocampus and to investigate how these changes related to the genetic basis of neurodevelopmental phenotypes. BL/6J mice were exposed to post-weaning SI (PD21-25) or treated as group-housed controls (n = 7–8 per group). RNA sequencing was performed on tissue samples from the hippocampus of adult male and female mice. Four hundred and 1,215 differentially-expressed genes (DEGs) at a false discovery rate of < 0.05 were detected between SI and control samples for males and females respectively. DEGS for both males and females were significantly overrepresented in gene ontologies related to synaptic structure and function, especially the post-synapse. DEGs were enriched for common variant (SNP) heritability in humans that contributes to risk of neuropsychiatric disorders (schizophrenia, bipolar disorder) and to cognitive function. DEGs were also enriched for genes harbouring rare de novo variants that contribute to autism spectrum disorder and other developmental disorders. Finally, cell type analysis revealed populations of hippocampal astrocytes that were enriched for DEGs, indicating effects in these cell types as well as neurons. Overall, these data suggest a convergence between genes dysregulated by the SI stressor in the mouse and genes associated with neurodevelopmental disorders and cognitive phenotypes in humans.

Introduction

Early life stress (ELS) includes childhood exposure to a range of adversities, and is associated with increased risk for neurodevelopmental disorders [1]. One such stressor is social isolation (SI), a situation in which an individual is deprived of typical and expected social interaction. These interactions are fundamental to normal social development [2] and exposure to SI during vulnerable periods of neurodevelopment can impact on both the behaviour and neurobiology of those affected [3, 4]. The detriment of SI-induced changes is clear, with evidence that exposure contributes to increased risk of anxiety and depression [5, 6], schizophrenia [7] and cognitive decline [8].

The use of rodent models of SI to investigate behaviour is common. Mice exposed to post-weaning SI tend to experience increased anxiety [9–16], increased depressive-like behaviour [11, 13], decreased cognitive ability [13, 14, 17–19], increased aggression [12, 20] and decreased sociability [10, 21] when compared to group-housed controls. Some of the neurobiology relevant to SI-induced behaviour in rodents has been mapped [22]. Targeted analyses have implicated neuronal growth factors such as BDNF [13, 16, 17, 20], hormones such as oxytocin [23], inflammatory mediators such as cytokines [24–26] and the function of most main neurotransmitter systems [22] as being involved in behaviours found in SI-exposed rodents. Rodents subjected to SI display dysregulation of genes crucial to the function of glutamatergic [14, 27, 28], GABAergic [28–30], dopaminergic [31] neurotransmission, all of which are systems thought to have a part to play in psychiatric disorders [32].

To our knowledge, no mouse studies of SI have included full transcriptome gene expression analysis of the molecular changes caused by SI in the hippocampus. Previously, transcriptomic analysis of the basolateral amygdala in socially-isolated mice identified genes associated with aggressive behaviour and found evidence of upregulated ion channel function, while genes related to limbic system development and cognition were downregulated. Furthermore, in the ventral tegmental area, genes related to neuropeptide signalling were downregulated and genes related to synaptic signalling were upregulated [33]. Other transcriptomic work using microarray in rat cortex [29] also found dysregulation of genes related to synaptic structure and function, particularly in relation to inhibitory GABAergic synapses.

Previous work by our group [10] showed that mice exposed to post-weaning SI exhibited a significant increase in anxiety related behaviours (males and females) and decreased sociability (females) compared to animals that were group-housed. We now extend our previous investigation by performing RNA sequencing (RNA-seq) to identify differentially expressed genes (DEGs) in the brains of the same animals–specifically in the hippocampus, a region that regulates stress response and emotion [34, 35]. Transcriptomic changes were found to be induced in both male and female hippocampus by SI. These differentially expressed genes (DEGs) encoded protein involved in synaptic structure and function. We further considered how these transcriptomic changes converge with the biological basis of human psychiatric disorders and behaviours by investigating if the DEGs were enriched for common heritability contributing to neurodevelopmental phenotypes and enriched for genes harbouring rare de novo variants contributing to neurodevelopmental disorders. Finally, we explored which specific cell types were enriched for DEGs induced by SI.

Materials and methods

Ethics statement

All procedures received ethical approval from the local Animal Care Research Ethics Committee (ACREC) and the Health Products Regulatory Authority in Ireland (licence number AE19125/P083).

Social isolation procedure

Tissues used in this analysis were obtained directly from the animals used in our previous study [10], consisting of post-weaning (P21-25) adult male and female C57 BL/6J mice (Charles River Laboratories, UK) assigned to either group-housed cages (3–4 animals per cage) or single housed cages (SI; n = 7–8 per group) until animals were sacrificed by brief exposure to CO2 (90s) followed by immediate decapitation after a 60 day period. Whole brain was dissected on ice and hippocampus was snap-frozen in dry-ice before being stored at −80 °C.

Sample preparation

Total RNA was extracted from frozen hippocampus using the Absolutely RNA Miniprep kit (Agilent; product code: #400800) following appropriate standard procedure for quantity of tissue. Once purity and integrity (RIN > 6) was confirmed, isolated RNA-seq was performed on the Illumina NovaSeq (paired-end; 2x150 bp reads) sequencing platform producing a minimum of > 20M reads/sample (Genewiz Germany GmbH).

Differential expression analysis

Raw data was received through SFTP in FASTQC format. Trimmomatic v0.39 [36] was used to remove low quality and adapter sequences from the paired-end reads (LEADING:3, TRAILING:3, MINLEN:36). Salmon v1.8.0 [37] was used to quasi-map and quantify reads. The DEseq2 v1.24.0 [38] R package was used to test genes for differential expression. The sva v3.32.1 svaseq [39] function was used to detect batch effects in individual groups. Significant surrogate variable (SVs) were introduced into the differential expression model to control for technical batch effects. DEGs were defined at a false discovery rate (FDR) of < 0.05. Fold changes were shrunk using apeglm v1.6.0 [40]. Genes were converted to human orthologues using biomaRt v2.40.5 [41] where necessary.

Gene ontology analysis

Gene ontology (GO) analysis was done using ConsensusPathDB (http://cpdb.molgen.mpg.de/) [42] over-representation analysis. Ontologies with GO term levels 2–5 were tested and ontologies with a FDR-corrected p-value < 0.05 were considered significantly enriched. In order to limit GO analysis to ontologies relevant to the synapse, sets of DEGs also tested for enrichment using SynGO (https://www.syngoportal.org/) [43], an expert-curated resource for synaptic GO analysis. Ontologies with an FDR corrected p-value < 0.05 were considered significantly enriched.

Testing genes for enrichment of common genetic risk variants associated with neurodevelopmental phenotypes

Data on common variants (SNPs) associated with human phenotypes were accessed in the form of genome-wide association study (GWAS) summary stats for a range of phenotypes relevant to neurodevelopment and psychiatric disorders including schizophrenia (SCZ; GWAS based on 67,390 cases and 94,015 controls) [44], intelligence (IQ; 269,867 individuals) [45], educational attainment (EA; 766,345 individuals) [46], bipolar disorder (BPD; 41,917 cases and 371,549 controls) [47], major depressive disorder (MDD; 246,636 cases and 561,190 controls) [48] and anxiety-tension (Anx-Ten; 270,059 individuals) [49]. As control phenotypes, GWAS data for three brain-related disorders: Attention deficit/hyperactivity disorder (ADHD; 20,183 cases and 35,191 controls) [50], Alzheimer’s disease (AlzD; 71,880 cases and 383,378 controls) [51], stroke (40,585 cases and 406,111 controls) [52] and two non-brain related disorders: type-2 diabetes (T2D; 74,124 cases and 824,006 controls) [53] and coronary artery disease (CAD; 22,233 cases and 64,762 controls) [54] were used. Stratified LD Score regression (sLDSC) was used to investigate if gene-sets were significantly enriched for SNP heritability contributing to the test and control phenotypes [55, 56]. Gene start and stop coordinates of each DEG on GRCh37 were found using biomaRt v2.40.5 [41]. Annotation files were generated for each chromosome in each set of DEGs using 1000 genomes European cohort SNPs and a window size of 100kb extended to coordinates [57]. LD scores were estimated within a 1cM window using 1000 Genomes Phase 3 European reference panel. Heritability was stratified in a joint analysis between 53 previous function genomic annotations [56] and each set of DEGs. Only SNPs from HapMap Project phase 3 SNPs with a MAF > 0.05 were considered in this analysis.

For phenotypes with significantly enriched heritability from the LDSC analysis, MAGMA [58] was used to test individual genes for association. First, we annotated the SNP data to genes using the build 37 gene locations (https://ctg.cncr.nl/software/MAGMA/aux_files/NCBI37.3.zip) and 1000 Genomes European Panel reference (https://ctg.cncr.nl/software/MAGMA/ref_data/g1000_eur.zip) files, the latter which MAGMA uses to account for linkage disequilibrium (LD) between SNPs. Second, MAGMA generated p-values for individual gene reflecting their level of association with the test and control phenotypes. Genes with a Bonferroni-corrected p-value of < 0.05 (correcting for the number of genes tested) were considered genome-wide significant for association with each phenotype.

Testing genes for enrichment of de novo mutations reported in neurodevelopmental disorders

Rare de novo mutations (DNMs) contributing to a phenotype can be detected using exome sequencing of trios including an affected proband and their biological parents. To test if DEGs were enriched for DNMs that contribute to neurodevelopmental disorders, we analysed DNMs reported in studies of autism spectrum disorder (ASD), intellectual disability (ID), SCZ and developmental disorders (DD). The functional class and gene location of DNMs identified in patients with ASD (n = 6,430), ID (n = 192) and in unaffected siblings (n = 1,995) and controls (n = 54) based on exome sequencing of trios were sourced from [59] and [60]. Genes harbouring DNMs reported in affected SCZ trios (n = 3,394) were taken from [61] and [62]. DNMs identified in developmental disorders (DD) were sourced from [63]. Mutations used for DD were subject to additional filtering based on posterior probability of de novo mutations, as described in [63]. To account for underlying mutational burden associated with the test phenotypes, results were then subject to a competitive test against background de novo mutation rate using a two-sample Poisson rate ratio test. Results with a Bonferroni-adjusted p-value of < 0.05 (correcting for the number of mutational classes and disorders tested) were considered significantly enriched.

Cell type enrichment

Data from single cell RNA-seq (scRNA-seq) of the mouse brain (565 cell types) [64] was used to test if different cell types were enriched for DEGs. Analysis was performed using the expression-weighted cell type enrichment (EWCE) R package [65], which investigated whether the cell types were significantly enriched for a gene-set when weighted by gene expression. Cell types were considered significantly enriched at a Bonferroni-corrected p-value of < 0.05 (correcting for the number of cell types tested).

Results

Differential gene expression

Table 1 summarises the numbers of DEGs induced by SI detected in male and female hippocampal tissue at FDR < 0.05. In total, 1,215 significant DEGs were identified in females and 400 in males. In both sexes, approximately twice as many DEGs were found to be downregulated than upregulated. Among the downregulated DEGs, 98 were common between males and females. Of the upregulated DEGs, 30 were common between males and females. Expression log 2 fold change (Log2FC) was highly consistent among DEGs shared between male and female samples, producing a R2 correlation coefficient of 0.88 (p-value < 2.2e-16). Full differential expression results can be found in S1 Table.

Table 1. Summary of DEGs induced by SI in hippocampus.

Set DEGs at FDR < 0.05 Downregulated Upregulated
Female 1215 810 (67.7%) 405 (33.3%)
Male 400 259 (64.7%) 141 (35.3%)
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GO and SynGO analysis

GO analysis was performed in order to gain insight into the functional roles of DEGs. ConsensusPathDB was used to identify GO biological processes (BP), cellular compartments (CC) and molecular functions (MF) terms enriched for DEGs. A total of 738 GO terms were found to be enriched for the female DEGs and 306 GO terms were enriched for the male DEGs at FDR < 0.05. (S2 Table). Of these enriched terms, 72 (9.8%) of the female and 36 (11.8%) of the male contained any of "neuro", "synap", "dendr" or "axon", indicating their relation to having neuronal, axonal or synaptic structure or function. Female DEGs were highly enriched for a number of neurodevelopmental GO terms. The most enriched level 4 and level 5 terms were nervous system development (GO:0007399, FDR-adjusted p-value = q-value = 2.76E-16) neurogenesis (GO:0022008, q-value = 9.02E-16), neuron development (GO:0048666, q-value = 1.26E-15), generation of neurons (GO:0048699, q-value = 1.57E-15), neuron projection development (GO:0031175, q-value = 1.57E-15) and neuron differentiation (GO:0030182, q-value = 2.57E-15). The significantly enriched GO terms for the male DEGs included nervous system development (GO:0007399, q-value = 6.79E-6), neuron part (GO:0097458, q-value = 9.82E-5), central nervous system development (GO:0007417, q-value = 3.31e-4) and brain development (GO:0007420, q-value = 4.61e-4).

Following up these neuronal GO term enrichments, additional GO analysis was performed using SynGO [43] to gain insight into the synaptic CCs and BPs enriched for DEGs (Fig 1; S3 Table). All enriched SynGO analysis terms were related to presynaptic and postsynaptic structure (CC) and function (BP) and provide greater focus on the precise neurobiological changes brought about by SI. The female DEG set showed significant enrichments in 39 SynGO terms. Both postsynapse (GO:0098794, q-value = 5.14e-17) and presynapse (GO:0098793, q-value = 3.15e-4) CCs were significantly enriched as well as a number of specific postsynaptic ontologies including postsynaptic density (GO:0014069, q-value = 2.25e-8), postsynaptic specialisation (GO:0099572, q-value = 1.56e-7), translation at postsynapse (GO:0140242, q-value = 5.76e-4) and postsynaptic organisiation (GO:0099173, q-value = 2.19e-3). The male DEGs showed significant enrichment in the postsynapse (GO:0098794, q-value = 3.67e-3) but not presynapse CCs. Similar to that of the females, male DEGs were also significantly enriched for processes of postsynaptic specialisation (GO:0099572, q-value = 0.003667) and postsynaptic density (GO:0014069, q-value = 0.012).

Fig 1. SynGO cellular compartment (CC) enrichments of female and male DEG sets.

Fig 1

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Annotated sunburst of enriched SynGO synaptic CC ontologies. Enrichments were detected for both pre and post-synaptic terms, however most significantly-enriched ontologies are post-synaptic. DEGs from females showed more enrichments in SynGO terms than DEGs from males.

Enrichment for genes contributing to human neurodevelopmental phenotypes

To assess whether genes dysregulated in mouse hippocampal tissue by SI harboured genetic risk for human neurodevelopmental phenotypes, enrichments for common heritability (SNPs) and genes harbouring rare DNMs contributing to neurodevelopmental disorders and related phenotypes were investigated. sLDSC [55, 56] was used to test common SNPs. The 1,215 female DEGs accounted for 9.5% of the total SNPs in the analysis, while the smaller male set of 400 DEGs accounted for 3.5% of total SNPs. Results can been seen in Table 2, showing percentage of heritability (% h2) and enrichment p-value in SNP-based heritability for SCZ, EA, IQ and BPD for the female DEG set and enrichments in SCZ, IQ and BPD for the male DEG set. Of the five control phenotypes tested, just one (coronary artery disease) showed a marginal enrichment for the female DEG set. Full results from the sLDSC analysis can be found in S4 Table.

Table 2. Enrichment of SNP-based heritability for test phenotypes.

DEG Set % of Total SNPs SCZ IQ EA MDD BPD Anx-Ten
% h2 P* % h2 P % h2 P % h2 P % h2 P % h2 P
Female 9.5% 13.3% 2.27E-05 12.9% 0.000109 13.3% 2.60E-07 11.7% 0.0472 14.2% 0.000388 12.3% 0.0422
Male 3.5% 6.2% 7.08E-05 5.0% 0.00276 4.5% 0.0132 4.6% 0.0434 7.2% 2.51E-05 4.1% 0.4202
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Results in bold survive Bonferroni correction for multiple testing (including control phenotypes)

Given there was evidence of convergence of SI-induced DEGs and genes contributing to risk of neurodevelopmental phenotypes, we investigated which genes may be driving this connection. For the test phenotypes with significant enrichments in LDSC (SCZ, EA, IQ and BPD), MAGMA [58] was used to test which genes had genome-wide significant associations. Genes were considered significantly enriched at a Bonferroni-corrected p-value of < 0.05. A total of 619 genes were genome-wide significant for SCZ, 160 for BPD, 377 for IQ and 689 for EA. Full MAGMA gene-based analysis results can be found in S5 Table. These lists were further restricted to DEGs that were common between male and female hippocampus and with consistent direction of expression change. A total of ten genes, shown in Table 3 below, matched these criteria and were significantly associated with one or more of SCZ, BPD, IQ or EA. Three of the 10 highlighted genes were genome-wide significant for two phenotypes, while 7 had a single significant gene-phenotype association from MAGMA. At least one gene was associated with SCZ, BPD, IQ or EA. All ten genes were downregulated, with male hippocampus consistently showing greater expression change.

Table 3. Genes found to be common between DEG analysis (same direction of effect in females and males) and MAGMA gene-based association analysis.

Symbol Gene Name Associated Phenotypes Direction in Females Direction in Males
RIMS1 Regulating Synaptic Membrane Exocytosis 1 SCZ, BPD Downregulated, Log2FC = -0.22 Downregulated, Log2FC = -0.32
ZNF365 Zinc Finger Protein 365 SCZ, BPD Downregulated, Log2FC = -0.21 Downregulated, Log2FC = -0.32
AGAP1 ArfGAP With GTPase Domain, Ankyrin Repeat And PH Domain IQ, EA Downregulated, Log2FC = -0.21 Downregulated, Log2FC = -0.25
TTBK1 Tau Tubulin Kinase 1 SCZ Downregulated, Log2FC = -0.14 Downregulated, Log2FC = -0.31
PPP1R16B Protein Phosphatase 1 Regulatory Subunit 16B SCZ Downregulated, Log2FC = -0.18 Downregulated, Log2FC = -0.31
SPTBN2 Spectrin Beta, Non-Erythrocytic 2 BPD Downregulated, Log2FC = -0.21 Downregulated, Log2FC = -0.32
SHANK2 SH3 And Multiple Ankyrin Repeat Domains 2 BPD Downregulated, Log2FC = -0.14 Downregulated, Log2FC = -0.27
UTRN Utrophin EA Downregulated, Log2FC = -0.25 Downregulated, Log2FC = -0.24
PHF2 PHD Finger Protein 2 IQ Downregulated, Log2FC = -0.12 Downregulated, Log2FC = -0.18
ZBTB4 Zinc Finger And BTB Domain Containing 4 EA Downregulated, Log2FC = -0.13 Downregulated, Log2FC = -0.33
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Using gene-level data from these studies, the R package denovolyzeR [66] was used to test gene-sets for enrichment for genes harbouring rare DNMs contributing to SCZ, ASD, ID and DD. Categories of synonymous (syn), missense (mis) and loss-of-function (lof) mutations were considered for analysis. Following a competitive analyses, gene-sets were considered enriched at a Bonferroni-corrected p-value of <0.05. Table 4 shows enriched categories of variants in each gene-set. Female DEGs showed strong enrichment for genes containing missense variants contributing to DD. Male DEGs were also enriched for missense variants contributing to DD as well as being enriched for genes containing loss-of-function mutations contributing to DD and ASD. As a control, gene-sets were not enriched for synonymous variants in any of the disorders. No enrichments were seen in trios containing unaffected siblings or controls. Complete results from rare variant analysis can be found in S6 Table.

Table 4. Enrichments for genes harbouring rare de novo mutations in male and female gene sets.

Set DEGs SCZ
n = 3394 Trios		 ASD
n = 6430 Trios		 ID
n = 192 Trios		 DD
n = 4293 Trios
Female Hippocampus 1215 ns ns ns mis (p = 5.20e-6)
Male Hippocampus 400 ns lof (p = 2.60e-5) ns lof (p = 8.23e-8)
mis (p = 8.05e-5)
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SCZ = Schizophrenia; ASD = Autism; ID = Intellectual Disability; DD = Developmental Disorder

ns = non-significant; lof = loss of function mutations; mis = missense mutations; Results in bold survive Bonferroni correction for multiple testing

Cell-type enrichment analysis

Cell types enriched for male and female DEGs were tested using EWCE [65]. Both sets were tested in scRNA-seq gene expression data from mouse brain [64]. Although this dataset contains expression data on 565 cell types, enrichments from this analysis was restricted to cell types from the hippocampus (n = 104). Cell types were considered enriched at a Bonferroni-corrected p-value of < 0.05. The primary cell type found to be enrichment in female DEGs was of glial origin. Of the cell types in the mouse brain data [64] three hippocampal cell types, HC_7–1 (p < 0.00001), HC_7–2 (p < 0.00001) and HC_7–3 (p < 0.00001) were enriched; all three were populations of hippocampal astrocytes. No hippocampal cell types were considered significantly enriched after multiple testing correction in the male DEG set. Full cell type enrichment results found in S7 Table.

Discussion

This study builds on our previous behavioural work [10] using post-weaning SI mice and here investigates the molecular consequences of the environmental stressor on the mouse brain. The use of RNA-seq to investigate the transcriptomic changes in mouse hippocampus caused by SI presents a novel insight into the underlying molecular underpinnings of altered behaviour. Furthermore, we used data from studies of rare and common risk variants for neurodevelopmental phenotypes to investigate the relevance of the SI model to human illness and behaviour.

Differential expression analysis detected hundreds of dysregulated genes with approximately three times the number of DEGs in female (n = 1,215) compared to male (n = 400) hippocampal tissue. This is consistent with our behavioural data in these animals [10], which showed females to be more susceptible to SI-induced behavioural measures of anxiety and sociability. GO analysis identified that DEGs in both males and females were enriched in structural and functional synaptic ontologies with DEGs from females implicated in both overall presynapse and postsynapse ontologies, while DEGs from males were only implicated postsynapse ontologies. In the context of our behavioural data [10], this suggests that presynaptic processes may play a role in facilitating SI-induced behaviours of anxiety and decreased sociability observed in the females. Synaptic biology is implicated in many neurodevelopmental disorders. In SCZ, the latest GWAS implicates genes involved in the organisation, differentiation and function of synapses [44]. For ASD, rare variants in genes involved in synapse structure including from the SHANK [67], NRXN [68] and NLGN [69] gene families are linked with the disorder.

In our rare variant analysis, enrichment for genes containing loss-of-function mutations contributing to ASD were found in the DEGs from males but not females. DEGs from both males and females were enriched for genes harbouring DNMs contributing to DD, however the DEGs from males showed a greater level of enrichment. Our sLDSC analysis detected enrichments in SNP heritability associated with SCZ, BPD and IQ in both male and female DEG sets with again males displaying stronger enrichments in all of these phenotypes. Together, the rare variant and heritability analyses indicate that the set of DEGs from male SI mice, despite only totalling a third of the number of DEGs in the set from female SI mice, converge more strongly with the genes that underpin risk for neurodevelopmental disorders and associated phenotypes in humans.

Anxiety was the most prominent phenotype induced by SI in our behavioural work [10]. Although a nominal enrichment in heritability was seen in the Anx-Ten phenotype for the DEGs from females (p = 0.042), this result did not survive multiple-testing correction. However, it is important to note that anxiety disorders are under less genetic influence (heritability of 20–60%) [70] than for example SCZ and BPD (both with heritability of 60–80% heritable) [44, 47]. As a result and despite a very large sample size, the GWAS of Anx-Ten identified just fourteen independent loci [49] and therefore we were unlikely to detect that our sets of DEGs were strongly enriched for common variants associated with this phenotype. Based on the pretext that no SI-induced cognitive changes were detected in these mice [10], the enrichment in IQ and EA heritability in the set of DEGs from female mice suggests that a subclinical effect of SI on cognition may have been present in this circumstance.

Ten genes were prioritised based on being consistently differentially expressed in males and females, as well as having genome-wide significant associations with any of the phenotypes that were enriched in the sLDSC analysis. Three genes, RIMS1, ZNF365 and AGAP1, were associated with two of the four phenotypes tested. RIMS1 plays an important role regulating and localising calcium channels and neurotransmission [71–75], regulating synaptic plasticity [76–79] as well as being crucial for learning and memory in mouse [80]. In humans, RIMS1 is also an ASD candidate risk gene implicated in rare variant studies [81–83]. ZNF365 (also called DBZ) is involved in neurogenesis, especially regarding basket cells in the somatosensory cortex [84]. It is also involved in oligodendrocyte differentiation [85] and regulating dendritic spine density in pyramidal neurons [86]. ZNF365 interacts with the SCZ candidate gene, DISC1, which through its role on oligodendrocyte differentiation could be a contributor to SCZ and MDD [87]. AGAP1 (also called CENTG2) regulates dendritic spine morphology [88] and is involved in neurotransmitter release in dopaminergic neurons [89]. In humans, variation in AGAP1 has been associated with to ASD [90], SCZ [91]and ASD/ID [92]. Further human data from PsychEncode is consistent with these prioritised mouse DEGs, with RIMS1 downregulated in post-mortem brain samples SCZ (Log2FC = -0.05, FDR = 0.004) and ZNF365 downregulated in post-mortem brain samples of ASD (Log2FC = -0.24, FDR = 0.002) [93].

Although GO analysis primarily implicated changes in synaptic biology caused by SI, the primary findings from cell type enrichment analysis were glial cells. Given the number of cell types analysed here, there is a strict Bonferroni threshold for cell types to be enriched (104 x 2 independent tests). Only cell types with a number highly unique genes overrepresented in our analysis will show up an enriched, which may explain the lack of neuronal cell types showing to be significantly enriched after correction. However, it is now well established that there is a non-neuronal component of neurodevelopment [94]. Astrocytes, enriched in this analysis, play a number of crucial functions to maintain a normal early developmental trajectory. These include maintaining a balanced extracellular environment, protecting neurons during neuroinflammation, and promoting synaptogenesis [95]. In the context of these results, altered expression of genes crucial to astrocyte function may impair the brain’s ability to deal with other biological effects of isolation, including neuroinflammation [96] and increased oxidative stress [97]. Human data also highlights the importance of astrocyte function. As discussed in Kruyer, Kalivas [98], human post-mortem data widely implicate morphological and molecular changes in multiple psychiatric disorders including SCZ, MDD and BPD discussed here.

Recent work in the SI field further summarises the effects of SI on the brain [4]. The authors reported overall changes in neuron biology caused by SI in rodents, including changes in neurogenesis, synaptogenesis, neurotransmission and cell morphology. Between GO results highlighting synaptic ontologies and prioritised genes being strongly implicated in neurotransmitter release, neurogenesis and cell neuron morphology, our findings generally align with these conclusions. Furthermore, there was an emphasis placed on the importance of glial cell types mediating the effects of SI [4]. This is also generally supported by our findings in two ways. First, the prioritised ZNF365 gene (downregulated in both males and females) plays a role in oligodendrocyte differentiation, which as discussed [4] is a process clearly playing an important role in dysfunction in an SI context. Second, we found that genes are dysregulated as a consequence of SI in hippocampal astrocytes, which as noted [4] are significantly activated as a result of early life SI in females [99].

There are a number of limitations to this present study. First, focusing on a single region limits the ability to generalise these results. Second, these data are generated from a stressor at a single timepoint. Investigating a number of developmental timepoints could help detect transient gene expression changes and could facilitate investigation into the impacts of earlier or later social stressors on gene expression in the mouse brain. Finally, we use a single technique to measure the effect of SI. Other techniques, especially epigenetic profiling using ATAC-seq, or the use of single-cell RNA-seq methods could add valuable context to gene expression profiles found in this data. Addressing these limitations in future studies could help shed light on the relationship between gene expression changes, altered behavioural phenotypes and known human risk variation for neurodevelopmental disorders.

In summary, we provide novel insight into how SI affects the mouse brain. The use of RNA-seq highlights gene expression changes related to synaptic biology that may underpin the changes in behaviour previously found in these animals. We show convergence of DEGs in socially isolated mice with genes containing genetic variation contributing to neurodevelopmental phenotypes in humans. We highlight genes such as RIMS1, ZNF365 and AGAP1 as candidates for studying disrupted developmental trajectories in disorders such as SCZ, BPD and ASD. Further, these data provide support for the molecular validity of the SI mouse model to study neurodevelopmental phenotypes with further behavioural, molecular and interventional investigations.

Supporting information

S1 Table. Differentially-expressed genes (DEGs) detected at FDR < 0.05.

(XLSX)

Click here for additional data file. (170.1KB, xlsx)
S2 Table. Full Gene Ontology (GO) results—All Ontologies (ConsensusPathDB).

(XLSX)

Click here for additional data file. (266.7KB, xlsx)
S3 Table. Full Gene Ontology (GO) results—Synaptic Ontologies (SynGO).

(XLSX)

Click here for additional data file. (37.9KB, xlsx)
S4 Table. Stratified Linkage Disequilibrium Score Regression (sLDSC) full results.

(XLSX)

Click here for additional data file. (12.1KB, xlsx)
S5 Table. MAGMA genome-wide significant genes.

(XLSX)

Click here for additional data file. (68.6KB, xlsx)
S6 Table. Full denovolyzeR results (competitive).

(XLSX)

Click here for additional data file. (12.5KB, xlsx)
S7 Table. Significantly-enriched hippocampal cell types in male and female DEGs.

(XLSX)

Click here for additional data file. (23.1KB, xlsx)

Data Availability

All relevant data are within the paper and its Supporting information files. Raw RNA-seq data is available under GEO accession number GSE246551.

Funding Statement

This work was funded by grants from the European Research Council (ERC-2015- STG-677467 to GD; https://erc.europa.eu/), Science Foundation Ireland (SFI- 16/ERCS/3787 to GD; https://www.sfi.ie/) and the Irish Research Council (GOIPG/2019/1932 to AL; https://research.ie/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Alexandra Kavushansky

19 Oct 2023

PONE-D-23-29486Social isolation-induced transcriptomic changes in mouse hippocampus impact the synapse and show convergence with human genetic risk for neurodevelopmental phenotypesPLOS ONE

Dear Dr. Morris,

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PLOS ONE

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Reviewer #1: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: Yes

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Reviewer #1: Yes

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Reviewer #1: This manuscript demonstrates how Post-weaning social isolation (SI) affects the expression of genes in mouse hippocampus and to investigate how these changes related to the genetic basis of neurodevelopmental phenotypes.

The final conclusion is drawn with "a convergence between genes dysregulated by　the SI stressor in the mouse and genes associated with　neurodevelopmental disorders and cognitive phenotypes in humans."

The study appropriately constructs hypotheses based on previous reports and carefully verifies them through experiments. The results of each experiment are appropriately discussed. The manuscript itself is logically and precisely written. Therefore, this paper is basically judged to be worthy of acceptance in this Journal. However, the following items should be reviewed before final decision.

The authors have reported in a previous study that mice exposed to post-weaning SI exhibited a significant increase in anxiety related behaviors (males and females) and decreased sociability (females) compared to animals that were group-housed. I would like to confirm whether behavioral disorders as a phenotype are also expressed under the present conditions.

In addition, please include a discussion of the possibility that any changes at the genetic level in animals subjected to post-weaning SI will always lead to behavioral disorders as a phenotype. In terms of genetic changes and phenotypic expression, is it possible to indicate specifically to what extent they are correlated? If so, please indicate it.

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Reviewer #1: No

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PLoS One. 2023 Dec 21;18(12):e0295855. doi: 10.1371/journal.pone.0295855.r002

Author response to Decision Letter 0

27 Oct 2023

Responses to Reviewer’s Comments:

1. This manuscript demonstrates how Post-weaning social isolation (SI) affects the expression of genes in mouse hippocampus and to investigate how these changes related to the genetic basis of neurodevelopmental phenotypes.

The final conclusion is drawn with "a convergence between genes dysregulated by the SI stressor in the mouse and genes associated with　neurodevelopmental disorders and cognitive phenotypes in humans."

The study appropriately constructs hypotheses based on previous reports and carefully verifies them through experiments. The results of each experiment are appropriately discussed. The manuscript itself is logically and precisely written. Therefore, this paper is basically judged to be worthy of acceptance in this Journal. However, the following items should be reviewed before final decision.

Response: We thank the reviewer for the positive comments regarding the study design, writing and discussion.

2. The authors have reported in a previous study that mice exposed to post-weaning SI exhibited a significant increase in anxiety related behaviors (males and females) and decreased sociability (females) compared to animals that were group-housed. I would like to confirm whether behavioral disorders as a phenotype are also expressed under the present conditions.

Response: The tissue used in this study is from a subgroup of the animals used in Desbonnet et al. (2022) in which the behavioural and immunological aspects of these animals were studied. This present study, which begun much later than the original study, assays gene expression in the hippocampus of the same socially-isolated and group-housed adult mice. The behavioural changes, summarised in Table 1 of Desbonnet et al. (2022) apply to these animals. Therefore, we can confirm the anxiety (males and females) and decreased sociability (females) phenotypes were expressed under the present conditions. The Materials and Methods states: “Tissues used in this analysis were obtained directly from the animals used in our previous study [Desbonnet et al. (2022)].”

3. In addition, please include a discussion of the possibility that any changes at the genetic level in animals subjected to post-weaning SI will always lead to behavioral disorders as a phenotype. In terms of genetic changes and phenotypic expression, is it possible to indicate specifically to what extent they are correlated? If so, please indicate it.

Response: This is an important point. First, we must consider the limitations of using a single timepoint (post-weaning), analyzing a single region (hippocampus) and using a single functional genomics technique (RNA-seq) in comparing genetic data and behavioural data. Although the hippocampus was chosen based on its established roles in stress response and emotion (Cameron and Glover 2015, Snyder et al. 2011), we may have missed out on behaviourally relevant region-specific expression changes in other parts of the mouse brain. Furthermore, by only using bulk RNA-seq, we may be missing out on important cell-type specific and epigenetic context to our data which could help more precisely link genetic changes and behaviour.

Regarding correlations, it is difficult to assess the true relationship between gene expression and behavioural changes in mouse models. Published studies will generally have a higher concordance of behavioural and molecular data. For example, of the targeted studies showing anxiety mentioned in the introduction section of this manuscript excluding Desbonnet et al. (2022), 6 out of 7 (86%) report on protein or gene expression and all of them show changes. Although it is certainly possible for gene expression changes to be present in the absence of significant behavioural changes e.g., an underpowered behavioural analysis, it’s highly unlikely that behavioural changes will be present in the absence of gene expression changes. Therefore, we generally consider behavioural changes a fundamental component to studies of social stress as it represents the strongest evidence of true response to the stressor.

Specific to our data, we are looking at global changes at a single timepoint without particular focus on individual genes. Therefore, a direct correlation between behavioural phenotypes and gene expression would be impractical. However, based on this comment, we could consider future more targeted studies (i.e. using lower throughput techniques such as qPCR) which focus on the specific relationship between gene expression and behaviour phenotype magnitudes. Furthermore, the incorporation of various developmental timepoints may further help understand and correlate these relationships.

Actions taken: We have added a paragraph in the discussion section, highlighting the limitations of this work. See below:

“There are a number of limitations to this present study. First, focusing on a single region limits the ability to generalise these results. Second, these data are generated from a stressor at a single timepoint. Investigating a number of developmental timepoints could help detect transient gene expression changes and could facilitate investigation into the impacts of earlier or later social stressors on gene expression in the mouse brain. Finally, we use a single technique to measure the effect of SI. Other techniques, especially epigenetic profiling using ATAC-seq, or the use of single-cell RNA-seq methods could add valuable context to gene expression profiles found in this data. Addressing these limitations in future studies could help shed light on the relationship between gene expression changes, altered behavioural phenotypes and known human risk variation for neurodevelopmental disorders.”

Cameron, H. A. and Glover, L. R. (2015) Adult neurogenesis: beyond learning and memory. Annu Rev Psychol, 66, pp. 53-81.

Desbonnet, L., Konkoth, A., Laighneach, A., McKernan, D., Holleran, L., McDonald, C., Morris, D. W., Donohoe, G. and Kelly, J. (2022) Dual hit mouse model to examine the long-term effects of maternal immune activation and post-weaning social isolation on schizophrenia endophenotypes. Behav Brain Res, 430, pp. 113930.

Koopmans, F., van Nierop, P., Andres-Alonso, M., Byrnes, A., Cijsouw, T., Coba, M. P., Cornelisse, L. N., Farrell, R. J., Goldschmidt, H. L., Howrigan, D. P., Hussain, N. K., Imig, C., de Jong, A. P. H., Jung, H., Kohansalnodehi, M., Kramarz, B., Lipstein, N., Lovering, R. C., MacGillavry, H., Mariano, V., Mi, H., Ninov, M., Osumi-Sutherland, D., Pielot, R., Smalla, K. H., Tang, H., Tashman, K., Toonen, R. F. G., Verpelli, C., Reig-Viader, R., Watanabe, K., van Weering, J., Achsel, T., Ashrafi, G., Asi, N., Brown, T. C., De Camilli, P., Feuermann, M., Foulger, R. E., Gaudet, P., Joglekar, A., Kanellopoulos, A., Malenka, R., Nicoll, R. A., Pulido, C., de Juan-Sanz, J., Sheng, M., Sudhof, T. C., Tilgner, H. U., Bagni, C., Bayes, A., Biederer, T., Brose, N., Chua, J. J. E., Dieterich, D. C., Gundelfinger, E. D., Hoogenraad, C., Huganir, R. L., Jahn, R., Kaeser, P. S., Kim, E., Kreutz, M. R., McPherson, P. S., Neale, B. M., O'Connor, V., Posthuma, D., Ryan, T. A., Sala, C., Feng, G., Hyman, S. E., Thomas, P. D., Smit, A. B. and Verhage, M. (2019) SynGO: An Evidence-Based, Expert-Curated Knowledge Base for the Synapse. Neuron, 103(2), pp. 217-234 e4.

Snyder, J. S., Soumier, A., Brewer, M., Pickel, J. and Cameron, H. A. (2011) Adult hippocampal neurogenesis buffers stress responses and depressive behaviour. Nature, 476(7361), pp. 458-61.

Decision Letter 1

Alexandra Kavushansky

30 Nov 2023

Social isolation-induced transcriptomic changes in mouse hippocampus impact the synapse and show convergence with human genetic risk for neurodevelopmental phenotypes

PONE-D-23-29486R1

Dear Dr. Morris,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Alexandra Kavushansky, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: (No Response)

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: (No Response)

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this revised manuscript, the authors have appropriately addressed the reviewers' comments, and the paper is considered acceptable to the Journal at this stage.

**********

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

**********

Acceptance letter

Alexandra Kavushansky

12 Dec 2023

PONE-D-23-29486R1

PLOS ONE

Dear Dr. Morris,

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    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Table. Differentially-expressed genes (DEGs) detected at FDR < 0.05.

    (XLSX)

    Click here for additional data file. (170.1KB, xlsx)
    S2 Table. Full Gene Ontology (GO) results—All Ontologies (ConsensusPathDB).

    (XLSX)

    Click here for additional data file. (266.7KB, xlsx)
    S3 Table. Full Gene Ontology (GO) results—Synaptic Ontologies (SynGO).

    (XLSX)

    Click here for additional data file. (37.9KB, xlsx)
    S4 Table. Stratified Linkage Disequilibrium Score Regression (sLDSC) full results.

    (XLSX)

    Click here for additional data file. (12.1KB, xlsx)
    S5 Table. MAGMA genome-wide significant genes.

    (XLSX)

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    S6 Table. Full denovolyzeR results (competitive).

    (XLSX)

    Click here for additional data file. (12.5KB, xlsx)
    S7 Table. Significantly-enriched hippocampal cell types in male and female DEGs.

    (XLSX)

    Click here for additional data file. (23.1KB, xlsx)

    Data Availability Statement

    All relevant data are within the paper and its Supporting information files. Raw RNA-seq data is available under GEO accession number GSE246551.

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