In the present study, we applied a multi-level approach involving methylomic, transcriptomic, and proteomic studies of rats’ brain tissues—FCx and Hp—aiming to identify factors and characterize their engagement in molecular pathways potentially altered in individuals prenatally treated with glucocorticoids. As previously described [25, 26], our research was conducted in an animal model of depression, the principle of which involves the administration of synthetic glucocorticoid (DEX) to pregnant females from the 14th day of pregnancy until delivery (see details in the “Materials and Methods” section). Prenatal exposure to DEX induces behavioral deficits expressed as the extended immobility time shown in the forced-swim test and anxiety behavior, a symptom common in depression, demonstrated by a decrease in the open arm entries and a reduction in the time spent in open arms in the elevated plus maze test as well as anhedonia [18, 25]. Moreover, our group also demonstrated that prenatal treatment with DEX generates deficits in memory processes (impairment of spatial and recognition memory) [26]. Nevertheless, the exact mechanism underlying these pathological consequences is still unclear. Epigenetic alterations are suspected as one of the possible mechanisms responsible for the long-lasting effects of adverse factors acting early in life, and some studies have already investigated this hypothesis (for review, see [42]).

In the current study, we revealed alterations in the methylation status of numerous genes, some of which are essential for the proper development and functioning of the brain. In particular, we focused on the CpG islands’ methylation status, which provides a more informative picture of the epigenetic regulation of gene expression compared to whole genes methylation level. Nevertheless, it should be noted that predicting the inherent modulatory effects of methylation changes (especially in non-promoter regions) on gene expression remains challenging. In our study, we identified 200 differentially methylated CpG islands in the FCx when comparing DEX-treated and control animals, and 200 in the Hp. Enrichment analysis revealed several pathways associated mostly with intracellular signal transduction and ligand-receptor interactions. In the FCx, among the most important glucocorticoid-driven effects was alteration in the cAMP signaling pathway. cAMP, 3′,5′-cyclic adenosine monophosphate, is generated from adenosine triphosphate (ATP) by G-proteins that are associated with metabotropic receptors and can be released upon receptor activation. cAMP is a crucial signaling molecule involved in various cellular processes in the central nervous system (CNS), regulating i.a. synaptic plasticity, neuronal growth and development, and memory consolidation as well as glucose and lipid metabolism [43]. Since it is known that metabolic disturbances in the brain may be responsible for the onset of depression and astrocytes play a pivotal role in the supplying of energy substrates to neurons, astrocytic cAMP may regulate glycogenolysis and lactate release, whose level was found to be elevated in the FCx of prenatally DEX-treated rats, as we have shown previously [25]. We also demonstrated that a reduction in the oxidative phosphorylation process and in consequence diminished ATP synthesis may result from the reduced transport of lactate from astrocytes to neurons [25], and, as we observed in the current research, it can also be the consequence of alterations in the methylation status of genes involved in the cAMP signaling pathway. Moreover, among the genes characterized by the upregulated methylation pattern was Tomm20 which encodes mitochondrial outer membrane protein, mitophagy, and mitochondria abundance marker. Also in the Hp, Tomm20 was among the 10 genes with the most hypermethylated CpG island, which may further confirm the important role of dysregulation of mitochondrial function in the brain in the pathogenesis of depression induced by an unfavorable factor acting in the perinatal period. Therefore, it also seems that DEX may epigenetically regulate mitochondrial proteins’ gene expression and thus influence brain bioenergetics and energy production processes, which was documented earlier by us [25, 26]. Additionally, the dysregulation of the cAMP/Ca2+ signaling pathway is under debate as to whether it may be the missing link between brain insulin resistance and cognitive dysfunctions. Although scarce, there is data supporting this hypothesis, (reviewed in [44]). Our results also indicate the association between abnormalities in the regulation of the cAMP level, insulin signaling pathway, and cognitive disturbances (what we observed also under the influence of DEX) [25, 26]. It seems that also the control of neurotransmitters/hormone release could be accomplished through modifications of the cAMP pathway. In the current study, we found deregulation of methylation pattern in five genes involved in cAMP signaling, including genes encoding G protein-coupled receptor GABA (B1)—Gabbr1 and Somatostatin—Sstr2; phosphodiesterase Pde4c; and ionotropic glutamate NMDA receptor subunits Grin2c, Grin3b. The balance between factors catalyzing the formation of cAMP (adenylate cyclase) and its degradation (phosphodiesterases) determines the level of cellular cAMP. This in turn regulates the activity of downstream pathways, which at the final steps leads to the activation of N-methyl-d-aspartate (NMDA) receptors, playing a critical role in, e.g., synaptic plasticity. Importantly, a significantly lower level of cAMP was found in the brains of patients suffering from depression, when compared to control, healthy individuals [45], while this effect was reversed after selective serotonin reuptake inhibitor (SSRI) treatment [46]. Glutamatergic neurotransmission dysfunction, especially by NMDA receptors and changed expression of NMDA receptor subtypes, which leads to impaired NMDA receptor-mediated intracellular signaling pathways, contributes to the pathophysiology of major depressive disorder [47]. We found that prenatal GC exposure affected the methylation of genes encoding glutamate receptors and their subunits (e.g., Grin2c, Grin3b, Grm5) in the FCx and Hp. It was shown that the cAMP pathway in astrocytes increases the expression of glutamate transporters, but on the other hand, acute cAMP increase may lead to downregulation of glutamate uptake by astrocytes [48]. In fact, glutamate receptors play a crucial role in many processes including synaptic development, plasticity, learning, and memory, as well as in synapse maturation and synaptogenesis throughout life [47] and any interference in their function may lead to the development of neurological or neuropsychiatric disorders. However, not only dysfunction in the glutamatergic excitatory system but also an imbalance in excitatory and inhibitory neurotransmission (in which γ-aminobutyric acid, GABA, is involved), which are reported in humans and animal models and display depressive-like symptoms [49], seems to be epigenetically regulated. GABAB receptors modulate the activity of voltage-dependent calcium channels and currents, and their activation results in the inhibition of these channels and suppression of neuronal activity. Targeting GABAB receptor activity has been identified as a valuable therapeutic approach in depression and anxiety since the antidepressant effect of GABAB receptor antagonists (i.e., ketamine, Ro-25–6981) was shown [50,51,52,53]. In our study, we found decreased methylation levels in the promoter region of the Gabbr1 gene. These changes indicate a possible cause through which the behavioral cognitive alterations (impairment of spatial and recognition memory) that we observed in the animal model of depression may occur [25, 26]. The fact that modulation of the cAMP pathway plays a significant role in regulating cognitive processes is strongly supported by our observation that this pathway was strongly downregulated also at the protein level in the Hp—the structure most closely related to memory and learning processes. Furthermore, in both studied brain areas, the most hypermethylated CpG island was enclosed to the gene Dnah17 encoding microtubule-associated motor protein complexes. In neurons, microtubule function is crucial for axon structure and transport of neurotransmitter receptors, synaptic vesicle precursors, and mRNAs over long distances. It is also needed for an adequate response to cellular and environmental stress and according to this, dysregulation of motor activity may affect diverse neuronal functions [54].

Moreover, in the Hp, we also observed the downregulation of the transcript, an immediate-early gene (IEG) encoding c-fos. In neurons, c-fos expression appears to be stimulated by cAMP and Ca2+ through the activation of the CREB/CRE complex. IEGs play a crucial role in the interactions between genes and the environment because they control the expression of a wide range of genes, allowing for a long-lasting and sustained adaptation by providing the molecular basis for a rapid and dynamic response to neuronal activity. IEGs are utilized as a marker to understand neuronal ensembles associated with the formation of certain memories; they are essential to facilitate memory learning and storage [55]. The exposition to a learning paradigm or induction of long-term potentiation (LTP) results in enhanced IEG expression right following the treatment [56].

The findings from the present study showed the altered methylation pattern in genes encoding channels contributing to the calcium signaling pathway in both examined brain areas. Calcium ions (Ca2+) play a fundamental role in neurotransmission by facilitating the release of neurotransmitters, influencing postsynaptic responses, participating in various intracellular signaling pathways, and contributing to the overall regulation of synaptic communication as well as participating in astrocyte-neuron signaling [57, 58]. Astrocytic Ca2+ elevation induces glutamate (but also ATP, GABA, and d-serine) release from astrocytes in a process called “gliotransmitter release,” while these gliotransmitters mediate further neuronal excitation or inhibition [59]. Furthermore, a very recent study concerns the role of the Orai1 calcium channel, whose gene Orai1 was also one of those differentially methylated in the Hp in our study. It was shown that deletion of this channel in astrocytes leads to decreased expression of genes involved in inflammation, metabolism, and cell cycle pathways, as well as reduction in cellular metabolites and ATP production [60]. In the Hp, loss of Orai1 attenuates inhibitory neurotransmission and inflammation-induced astrocyte Ca2+ signaling, while in Orai1 knockout mice amelioration of LPS-induced depression-like behaviors including anhedonia and helplessness were reported [60].

Enrichment analysis of differentially methylated regions in the FCx unclosed also Wnt and Hippo signaling pathways to be affected by prenatal DEX treatment. In the adult brain, increasing evidence suggests that the Wnt pathway plays an essential role in the regulation of structure and function of the nervous system, synaptic transmission, and neurogenesis, while dysregulation of this pathway has been associated with brain disorders, such as Alzheimer’s disease and mood disorders [61, 62]. Although the Wnt signaling pathway has been intensively studied, also in the context of excess glucocorticoid exposure, data concerning prenatal glucocorticoid exposition are sparse. It is known that knock-down of Wnt pathway-associated genes led to alterations in the Wnt/β-catenin signaling, neurogenesis deficits, and depression-like behavior, which was reversed upon Wnt overexpression [63]. Also, other studies point to the involvement of Wnt signaling in the regulation of new neuron development and cognitive function [64]. Furthermore, components of the Wnt pathway are transcriptional and downstream targets of the Hippo pathway; epigenetically induced overexpression of the Hippo pathway and disruption of cellular processes involved in learning and memory are associated with an increased risk of stress-related psychiatric disorders [65]. The Hippo signaling pathway has been described as a key regulator of tissue growth, which in the CNS regulates proliferation, differentiation, and regeneration of neurons, neural progenitor cells, and neural stem cells (NSCs), but also plays a role in synaptic development [65,66,67,68]. To the best of our knowledge, our study is the first that associates prenatal glucocorticoid treatment with alterations in the Hippo signaling pathway-related gene methylation in adulthood. Because it is still poorly investigated, this aspect should be addressed in further studies to establish an extended explanation for this finding.

What is important, in our study, in the FCx is that genes clustered in Tight junction terms, which are responsible for cell–cell adhesion complex formation between brain endothelial cells in order to establish a barrier to limit the free flow of molecules between the blood and brain, were hypermethylated. Disruption of the blood–brain barrier (BBB) tight junctions and dysregulation of the barrier integrity is a common pathology reported in major psychiatric disorders with the most severe effects observed in patients diagnosed with depression and schizophrenia [69]. Functional and structural changes in the BBB endothelium, loss and mislocalization of tight junction proteins, and reduced astrocyte coverage contribute to BBB breakdown during the depression. Impaired endothelial function and BBB disintegration lead to cerebral perfusion deficiencies, which causes brain injury as well as emotional and cognitive issues. Loss of cerebrovascular integrity and BBB degradation can lead to infiltration of immune cells, activation of glial cells, and the production of a variety of inflammatory mediators and ROS, resulting in neuroinflammation and neuron death (reviewed in [70]). Our study demonstrated that these detrimental effects can be induced already at the stage of epigenetic programming.

Enrichment analysis indicates also that in both examined brain structures, methylation patterns were changed in genes classified into group signaling pathways regulating pluripotency of stem cells. It is currently assumed that neurodifferentiation in the adult brain occurs in specific niches of the subventricular zone of the lateral ventricles and the subgranular zone of the dentate gyrus of the Hp and at a very low level in cortical neurons. Stem cells are generally considered to be glycolytic and in agreement with this also neural stem cells (NSCs) in the brain are considered to have predominantly glycolytic activity. During neurogenesis, NSCs proliferate to become neural progenitor cells (NPC) which turn into mature neurons. A variety of modifications occurred during this process, such as the proliferation and activation of mitochondria, and the switch from glycolytic to aerobic metabolism, which are needed to adapt to changing metabolic demands. The activation of stem cells coincides with changes in mitochondrial morphology and function, which are believed to be fundamental for the proper neurogenesis process (reviewed in [71]). Therefore, it seems that the serious metabolic alterations that we observe in the brain, in the studied model of depression, may have their basis at the level of epigenetic regulation which may contribute to the disturbances in the process of adult neurogenesis. Simultaneously, NSCs are highly influenced by monoaminergic neurotransmitters and cognitive deficits that accompany depression and may also be exacerbated by decreased neurogenesis.

Alterations in the methylation pattern of genes related to the identified signaling pathways in both FCx and/or Hp indicate a highly possible mechanism driving the long-term consequences of prenatal glucocorticoid exposure on the brain persisting into adulthood, but additional studies were required to better understand the machinery of these modifications. Therefore, to deliver additional insights into the studied glucocorticoids’ long-term effects, the next step of our study involved the evaluation of the global transcriptome level. As a result of the applied treatment, we found 271 differentially expressed transcripts in the FCx of adult rats, among which further analysis clearly showed a significant number of genes associated with large and small ribosomal subunit assembly. Ribosomes are principal components of the protein synthesis machinery but ribosomal dysregulation in the pathophysiology of depression has been identified just recently. A comparative transcriptomic study, involving brain tissue collected from postmortem subjects with diagnosed depression and from rodents exposed to chronic variable stress demonstrated, in line with our results, down-regulation of ribosomal protein genes (RPGs) in both—humans and mouse models [72]. Moreover, an in vitro study showed that deregulation of the ribosomal protein gene expression was a glucocorticoid-driven response to stress. It has been established that post-mitotic neurons possess vast amounts of ribosomes and depend on controlled translation to develop and maintain their phenotypic features, such as neurite and synaptic morphogenesis, and additionally synaptic plasticity. The downregulation of RPGs in the brain can result in reduced ribosome biosynthesis throughout the neurons, leading to a global decrease in translation and protein synthesis in these cells. Moreover, since ribosomes are involved in the synthesis of transmitter receptors, synaptic scaffolding proteins, and other regulatory factors, the observed changes may indirectly alter the neurotransmission process. In addition to the above-mentioned effects, downregulation of RPG expression may alter ribosome composition in specific cellular locations which can be observed as the removal, modification, or substitute of a few RPs. Such changes have the potential to produce specialized ribosomes, altering the translation of, e.g., synaptic proteins, in a compartment-specific way (reviewed in [73]).

In line with this are data collected from proteomic analysis. They suggest that in both brain structures, prenatal DEX administration triggers a long-term decrease in the expression of a significant number of proteins. Moreover, although most of the DEPs identified in this study were downregulated, we also found important results regarding proteins whose expression was upregulated as a result of prenatal glucocorticoid overexposure. A drastic increase, not only in the protein but also in the transcript level, was detected for the carbonic anhydrase type III (Ca3) (protein level fold change = 16.3, mRNA level logFC = 3.1). Carbonic anhydrases (CAs) are a family of zinc metalloenzymes that catalyze the reversible hydration/dehydration of CO2/HCO3 which makes them highly important during processes, which require maintaining the appropriate cellular pH level [74]. The catalytic activity of the Ca3 isoform for the CO2 hydration process is quite low, but recently it was shown that cytosolic CAs can be involved in acid–base balance, respiration, carbon dioxide, and ions transport, and increasing evidence suggests their involvement in the pathogenesis of various disorders [75]. Among the cytosolic CA isoforms, Ca3 is an enzyme that is mainly present in tissues characterized by a high oxygen consumption rate, such as skeletal muscle, liver, and brain but it possesses a different, yet unknown physiological function. Pharmacological studies (on topiramate and acetazolamide) indicate that CA inhibitors, which are already used in clinics, may impair memory and, on the other hand, administering CA activators to animal models improves learning and memory [76].

In addition to the above-mentioned signaling pathways that we showed to be altered at the protein level, we also demonstrated significant modifications in several others—relevant in the context of depressive-like changes—molecular pathways. Currently gaining great interest among researchers is the PD-1/PD-L1 pathway. The immune system inhibition by the interaction of the PD-1 receptor on the immune cells with PD-L1 expressed on the surface of cancer cells is known as one of the key mechanisms allowing cancer cells to elude the immune response and subsequently promote tumor growth through immune tolerance. So far, changes in checkpoint regulation have been also observed in other disorders such as brain tumors, Alzheimer’s disease, ischemic stroke, spinal cord injury, and multiple sclerosis, but to date, there is only a few data on the immune checkpoints (ICP) involvement in the course of neuropsychiatric disorders. It has been demonstrated that the development of learned helplessness in mice is caused by Th17 cells found in the Hp, which over-express PD-1 [77]. Moreover, it was found that aging in C57BL/6 J mice is linked to elevated PD-1 expression in FCx and hypothalamic microglial cells, which could potentially exacerbate emotional and motor discoordination associated with aging [78]. Furthermore, PD-L2 expression on monocytes was found to be lower in bipolar disorder patients compared to healthy controls, according to Wu et al. [79]. Also, our unpublished data shows that PD-L1 expression is significantly decreased in the rat model of treatment-resistant endogenous depression and model of depression based on prenatal stress procedure, in the same brain structures as we examined in this study. Therefore it seems to be justified to hypothesize that the interaction of the immune system and the nervous system takes place at the ICP level.

Finally, at the protein level, we found the downregulation of the cell adhesion molecule (CAMs) pathway in the FCx. This pathway plays a key role during the formation of tissues and organs, as well as higher-order functions of living organisms such as neuronal communication. Also, contact between synapses in the CNS during neurotransmission is a specific type of adhesion, which is facilitated by CAMs. Through protein–protein interaction signaling cascades, CAMs actively control the forming of new synapses and modify the functioning of existing synapses via cell-to-cell connections [80]. Synaptically localized adhesion molecules can affect synaptic development, dendritic spine formation, synaptic receptor function, and synaptic plasticity. There are four main families of CAMs in the brain—cadherins, integrins, selectins, and immunoglobulin superfamily of cell adhesion molecules (IgSF). Integrins regulate spine function by controlling receptor trafficking in a subunit-specific manner, while cadherins control the formation of spines and synapses in excitatory neurons. CAMs are not only responsible for changes in synapses and network connectivity but they are also associated with permanent changes in physical forces throughout time, causing changes in brain plasticity. Knocking down the gene encoding α-integrin in Drosophila melanogaster leads to inhibition in short-term olfactory learning, which demonstrates the impact of integrins on long-term plasticity. These factors can also regulate LTP by affecting the activity of NMDARs and AMPARs. Cadherins modulate synapse activity after NMDA activation. However, their action is required for LTP and spine expansion, but not for long-term depression (LTD), spine density, or morphology, indicating their role in synaptic plasticity. IgSFs also control synapses by crosslinking to NMDARs and CaMKII through postsynaptic scaffolds (reviewed in [81]). In our study, we also showed that the most downregulated protein in the FCx was Dnmbp (more than 5 × decrease), a scaffold protein regulating actin cytoskeleton and synaptic vesicle pools, which also can escalate potential changes in synapses. In connection with this, in this study we also found hypermethylation of CpG islands of the Icam5 gene, encoding protein expressed on the surface of neurons, displaying adhesion activity and impacting functional synapse formation. This proves that adhesion proteins (also involved in neuron-microglial cell interactions) play a role not only in the course of normal development but also in pathological conditions.