Bioinformatic analysis identifies potential key genes of epilepsy
Epilepsy Sphingolipids Inflammation Central nervous system Gene expression Gamma-aminobutyric acid Neutrophils Signaling networks
Zhongyan Zhao Chuansen Lu Yike Zhu Dan Huang
Background Epilepsy is one of the most common brain disorders worldwide. It is usually hard to be identified properly, and a third of patients are drug-resistant. Genes related to the progression and prognosis of epilepsy are particularly needed to be identified.   Methods In our study, we downloaded the Gene Expression Omnibus (GEO) microarray expression profiling dataset GSE143272. Differentially expressed genes (DEGs) with a fold change (FC) >1.2 and a P-value <0.05 were identified by GEO2R and grouped in male, female and overlapping DEGs. Functional enrichment analysis and Protein-Protein Interaction (PPI) network analysis were performed.   Results In total, 183 DEGs overlapped (77 ups and 106 downs), 302 DEGs (185 ups and 117 downs) in the male dataset, and 750 DEGs (464 ups and 286 downs) in the female dataset were obtained from the GSE143272 dataset. These DEGs were markedly enriched under various Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) terms. 16 following hub genes were identified based on PPI network analysis: ADCY7, C3AR1, DEGS1, CXCL1 in male-specific DEGs, TOLLIP, ORM1, ELANE, QPCT in female-specific DEGs and FCAR, CD3G, CLEC12A, MOSPD2, CD3D, ALDH3B1, GPR97, PLAUR in overlapping DEGs.   Conclusion This discovery-driven study may be useful to provide a novel insight into the diagnosis and treatment of epilepsy. However, more experiments are needed in the future to study the functional roles of these genes in epilepsy.
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4 Discussion

In this study, we performed bioinformatics analysis to search for the potential key genes associated with epilepsy. Male and female epilepsy were compared to healthy controls respectively on the hypothesis that epilepsy in different genders had different mechanisms. The results showed that 302 male-specific DEGs, 750 female-specific DEGs, and 183 overlapping DEGs were successfully identified. Those DEGs were put into multi-step bioinformatic functional annotations, including GO, KEGG, and PPI analysis.

We observed that male, female and overlapping DEGs took part in similar and overlapping biological processes significantly. Most of the biological processes were involved in inflammation and immune system defense response, for example, immune response, leukocyte mediated immunity, T cell activation, etc. In KEGG analysis, sphingolipid signaling pathway was showed upregulated in male patients but downregulated in female patients. It is well known that sphingolipids, such as ceramide, sphingosine, sphingosine-1-phosphate, sphingomyelin, and gangliosides, play an important role in the regulation of steroidogenesis [17]. The sex hormones, such as androgens, estrogens, and progestogens, are thought to influence sex differences in epilepsy [18, 19]. For example, it is suggested that progesterone has anticonvulsant effects, while estradiol has neuroprotective effects along with mild anticonvulsant effects [4]; and androgen can amplify sex differences in the expression of some epileptic disorders [18]. The observation in our study indicated that sphingolipid signaling pathway may have a complex relationship with epilepsy in sex differences.

Based on the PPI networks, 16 hub genes were identified, which were ADCY7, C3AR1, DEGS1, CXCL1 in male-specific DEGs, TOLLIP, ORM1, ELANE, QPCT in female-specific DEGs and FCAR, CD3G, CLEC12A, MOSPD2, CD3D, ALDH3B1, GPR97, PLAUR in overlapping DEGs, respectively.

ADCY7 encodes a membrane-bound adenylate cyclase (AC), which is one of the most ubiquitous signal transduction molecules that catalyzes the formation of cyclic adenosine monophosphate (AMP) from adenosine triphosphate (ATP) [20, 21]. It is indicated that in the central amygdala (CeA) AC7 plays an important role in the modulation of presynaptic gamma-aminobutyric acid (GABA) release. When responding to ethanol and corticotropin-releasing factor (CRF), AC7 increases cAMP signaling and activates protein kinase A cascade, resulting in the release of presynaptic vesicular GABA [22]. GABA, formed within GABAergic axon terminals and released into the synapse, acts on receptors GABAA and GABAB. As the main inhibitory neurotransmitter in the cerebral cortex and hippocampus, GABA maintains inhibitory tension to balance nerve excitation. When the balance is disrupted, seizures may occur. Therefore, GABA agonists suppress seizures, while GABA antagonists cause seizures [23]. As the localization of AC7 was observed in the hippocampus, cerebral cortex, cerebellum, caudate-putamen, and nucleus accumbens [24], ADCY7 involving in regulation of GABA may be correlated with epilepsy.

C3AR1 is a Protein Coding gene. The protein C3a Receptor 1 encoded by this gene is an orphan G protein-coupled receptor for C3a, which is a proinflammatory mediator released during activation of the complement system [25]. The complement pathway is critical in innate immunity [26]. In Alzheimer’s patients, the overexpression of C3 and C3aR1 increases with cognitive decline and Braak staging. It has been shown that loss of C3aR1 in mice causes the rescue of tau pathology and attenuation of neuroinflammation, synaptic deficits, and neurodegeneration. C3aR1 is a critical regulator in neuronal tau pathogenesis and mediating central nervous system (CNS) immune network, as its direct target is STAT3 (signal transducers and activators of transcription) [27]. More and more evidence reveals there are common underlying mechanism mechanisms associated with network hyperexcitability and cognitive decline [28, 29]. Studies have shown that in mouse models the increased abnormal tau and amyloid-β proteins may have a synergistic effect on the occurrence of epileptic seizures [30–32]. Hyperphosphorylation of Tau has been reported in epileptic patients with different forms [33–37] and in a range of animal models of epilepsy [38–40]. Focus on C3AR1 mediating tau pathology might represent a novel opportunity to research therapy for epilepsy.

DEGS1 encodes Delta 4-Desaturase, Sphingolipid 1, a member of the membrane fatty acid desaturase family which is responsible for inserting double bonds into specific positions in fatty acids. The related pathways of DEGS1 are sphingolipid signaling pathway and sphingolipid metabolism [41, 42]. In the nervous system, sphingolipids are pivotal constituents of myelin formation in glial cells, which can improve the efficiency and speed of action potentials. Perturbations of the sphingolipid metabolism can result in rearrangements in the plasma membrane, which has been associated with the development of various neurological disorders [43–46]. It has been reported that a variant in DEGS1 leads to a novel early-onset autosomal recessive complex neurological disease with Intelligent disability, progressive spastic paraplegia, scoliosis, and epilepsy. The DEGS1 variant encodes C4-dihydroceramide desaturase, which plays an important role in a pathway of ceramide/phospholipids synthesis [41, 47]. In this study, the present findings also revealed that DEGS1 was correlated with epilepsy.

CXCL1 gene encodes a member of the CXC subfamily of chemokines. The CXCL1 protein plays a pivotal role by recruiting and activating neutrophils in inflammation when signaling through the CXCR2 receptor [48]. It is being increasingly recognized that immunity and inflammatory processes in the brain contribute to the pathogenesis of epilepsy [49–51]. The study reported that CXCL1 concentrations increase significantly after seizure onset, which results in a strong induction of chemotactic response from brain cells that recruits circulating neutrophils to the injured brain tissues [52]. The activated neutrophils can exacerbate the initial injury by the damage to the surrounding healthy area [53]. Thus, CXCL1 may be a novel therapeutic target for epilepsy.

TOLLIP encodes the Toll-interacting protein (Tollip), which is present in a complex with the interleukin-1 receptor associated kinase (IRAK). With the activation of IL-1β, the Tollip–IRAK complex is recruited and disrupted [54]. The overexpression of Tollip can impair IL-1β-induced activation of NF-κB, indicating that Tollip is an inhibitory modulator in inflammatory signaling [54, 55]. The cerebral cortex may display the highest density of Tollip protein [56]. Therefore, Tollip may be a potential target to provide neuroprotective effects by reducing neuroinflammation in epilepsy [57].

ORM1, accounting for 75% of plasma ORM, is an inflammatory factor with multiple activities [58]. Plasma concentration of ORM increases under the control of various regulatory mediators, including inflammatory stimuli, such as glucocorticoids, tumor necrosis factor (TNF)-α, interleukin (IL)-1, IL-8, IL-11, IL-6, and IL-6 related cytokines [59–61]. ORM has been revealed to have effects on immunoregulation, such as decreasing the rolling, adhesion, and migration of neutrophils [62–64]. In the ORM family, ORM1 is the unique member that could be considered as an acute-phase protein. ORM1 regulates the inflammation by contributing both anti- and pro-inflammatory signals to cytokine-mediated feedback mechanisms activated by the acute-phase response [65]. What’s more, ORM can also enhance the functional integrity of the blood-brain barrier (BBB) [66]. The study showed that decreasing the ORM1 expression may be a possible mechanism for the aggravation of BBB damage [67], while the disruption of BBB may underlie the occurrence of seizures and epilepsy [68, 69]. This study indicated that ORM1 may play important role in epilepsy.

ELANE encodes neutrophil elastase (NE), which belongs to the family of serine proteases. It has been reported that the mutations in the gene usually cause Cyclic neutropenia (CyN) and severe congenital neutropenia (SCN) [70, 71]. As NE is involved in immune responses and widely regarded as a regulatory factor in degenerative and inflammatory diseases through proteolysis of collagen-IV and elastin [72, 73], ELANE may be also associated with epilepsy.

The QPCT gene encodes glutaminyl cyclase (QC). QC is an enzyme responsible for catalyzing the posttranslational modification of N-terminal glutamate to pyroglutamate in many neuroendocrine peptides, which renders the protein more susceptible to neurotoxic [74]. Amyloid-β (Aβ) deposits have been found to be a characteristic neuropathological feature of Alzheimer’s disease (AD) [75]. N-terminally modified Aβ, pyroglutamate-amyloid-β (pE3-Aβ), is a major component of Aβ deposits specific to human AD [76, 77]. pE3-Aβ is processed by QC and/or its isoenzymes (isoQC), formed by cyclization of truncated Aβ species, rapidly aggregates and initiates other Aβ aggregates [78–80]. The formation of large amounts of pE3-Aβ has been shown to be QC-dependent. Reducing QC-dependent post-translational pE3-Aβ formation rate can in turn reduce the number of neurotoxic Aβ species [81]. As it is reported that in the pathophysiology of epilepsy Aβ may play an important role [82–84], QPCT may also serve as a novel strategy for the treatment of epilepsy.

FCAR encodes FcαRI or CD89, which is expressed on cells of the myeloid lineage [85] and is a bifunctional inhibitory/activating receptor for the Fc region of Immunoglobulin A (IgA) [86]. On one hand, FcαRI plays an anti-inflammatory role when binding to monomeric IgA and inducing inhibitory ITAMi signaling. On the other hand, when cross-linking IgA immune complexes, FcαRI mediates pro-inflammatory function, activating immune cells and leading to the elimination of pathogens [87, 88]. Neutrophilic activation is beneficial for infection; however, overabundant IgA complexes can trigger severe tissue damage causing various autoimmune diseases. Achiron A et al. found that FCAR participates in the pathogenic pathways in MS [89], which indicated FCAR may be involved in the various inflammatory responses in CNS. Targeting FcαRI might serve as a novel therapeutic strategy for epilepsy.

CD3G and CD3D encode CD3γ and CD3δ, respectively. Both CD3γ and CD3δ are part of the T-cell receptor/CD3 complex (TCR/CD3 complex) [90], which is crucial for the development, activation, and differentiation of T cells [91]. The CD3D gene defect, which occurs early in life, leads to severe immune deficiency, making a person susceptible to infection [92]. However, the CD3G mutation results in a milder clinical phenotype that is primarily autoimmune [93–95]. The severe immune deficiency may cause intracranial infection, while autoimmunity in brain may lead to autoimmune encephalitis, both of which are correlated to epilepsy [96–99]. However, the role of CD3G and CD3D in CNS is still not clear.

CLEC12A is a C-type lectin receptor (CLR) and a Src homology region 2 domain-containing phosphatase 1 and 2 (SHP-1 and -2)-associated receptor, highly expressed on human dendritic cells (DCs). CLEC12A contains a single immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic tail. In response to chemokine CCL2, ITIM can associate with SHP-1 and SHP-2, involved in inhibitory signaling as a key molecule to deliver immature DCs to the CNS across the BBB [100]. It has been reported that DCs can initiate autoimmune demyelination and inflammation in CNS by presenting antigen to autoreactive myelin-specific T cells [101], while in the CLEC12A-KO mice the reduction of DC infiltration and demyelination was observed [102]. Thus, CLEC12A may be a promising target to inhibit seizures in brain.

MOSPD2 (motile sperm domain-containing protein 2) is the surface protein predominantly expressed on cytoplasmic membrane of human monocytes. MOSPD2 is also found in neutrophils, but not in lymphocytes. It is revealed that MOSPD2 is critical in regulating the inflammatory monocyte and neutrophil migration without activating ligands [103]. The monocyte from blood can rise macrophage in CNS, which is dominant in demyelination [104]. More and more researches suggest that chronic demyelination in multiple sclerosis can induce seizure [105–107]. As it has been demonstrated silencing or neutralizing MOSPD2 not only reduced the proportion of inflammatory monocytes in the blood significantly but also inhibited monocyte migrating into CNS [108], MOSPD2 may be a potential target for the treatment of epilepsy.

ALDH3B1 encodes the protein that belongs to the ALDH3 protein family (Aldehyde Dehydrogenase Family 3) [109–111]. It has been shown that ALDH3B1 plays a critical role in the cellular defense against oxidative stress processes and aldehyde toxicity [112, 113]. Oxidative stress toxicity and lowered antioxidant defense are considered as contributing factors in the genesis and progression of epilepsy [114–116], while epileptic seizures, especially recurrent seizures may also increase oxidative stress, which will result in treatment resistance [117–120]. Oxidative stress can lead to the occurrence of lipid peroxidation (LPO) and resulting in plenty of aldehydes, such as 4-hydroxy-2-nonenal (4HNE) [121]. In the CNS, dopamine is metabolized to 3,4-dihydroxyphenylacetaldehyde (DOPAL), while both norepinephrine and epinephrine are metabolized to 3,4-dihydroxyphenylglycol aldehyde (DOPEGAL) [122]. Aldehydes, including DOPAL, DOPEGAL, and 4HNE, are neurotoxic and involving in Parkinson’s disease (PD) and AD [123, 124]. ALDH7A1, the other member of the ALDH protein family, has been demonstrated to be related to pyridoxine-dependent epilepsy [125–127]. Thus, it is indicated that ALDH3B1 may have a protective role in various brain diseases including epilepsy.

GPR97 belongs to the G protein-coupled receptors (GPCRs), the largest receptor superfamily broadly involved in the regulation of biological processes and various diseases, including CNS disorders, such as anxiety, depression, schizophrenia, epilepsy, Alzheimer’s disease, and Parkinson’s disease [128, 129]. GPR97, expressed in immune cells and lymphatic endothelial cells [130, 131], contributes to macrophage-associated inflammation [132]. GPR97 also regulates the development of B-cell and NF-κB activity [133], which plays a critical role in encephalitogenic T cell activation [134]. The study reveals that the loss of GPR97 results in the increase of constitutive expression and activation of NF-κB pathway components, in turn causing severe inflammation and demyelination in CNS [135]. Thus, modulation of GPR97 functions or its pathway may be a potential treatment of epilepsy.

PLAUR encodes urokinase-type plasminogen activator receptor (uPAR) [136], which is a glycoprotein linked to the cell membrane by a glycosylphosphatidylinositol anchor [137]. The uPAR is a key regulator in many processes involving in not only cell signaling, proliferation, differentiation, and migration, but also tissue remodeling [137–139]. uPAR plays an important role in the early and injured brain. The uPA-uPAR complex induces axonal growth and regeneration by stimulating neuronal migration and neuritogenesis via both proteolytic and nonproteolytic events [140–142]. The dysregulation of uPA/uPAR axis is involved in various CNS disorders [139]. In rats undergoing seizures, expression of uPAR is increased in interneurons [143], while deficiency of both uPA and uPAR in mice increases seizure susceptibility [143–145]. In this study, the present findings also revealed that PLAUR was correlated with epilepsy.