The Contributions of Thrombospondin-1 to Epilepsy Formation

Abstract

Epilepsy is a neural network disorder caused by uncontrolled neuronal hyperexcitability induced by an imbalance between excitatory and inhibitory networks. Abnormal synaptogenesis plays a vital role in the formation of overexcited networks. Recent evidence has confirmed that thrombospondin-1 (TSP-1), mainly secreted by astrocytes, is a critical cytokine that regulates synaptogenesis during epileptogenesis. Furthermore, numerous studies have reported that TSP-1 is also involved in other processes, such as angiogenesis, neuroinflammation, and regulation of Ca²⁺ homeostasis, which are closely associated with the occurrence and development of epilepsy. In this review, we summarize the potential contributions of TSP-1 to epilepsy development.

Introduction

Epilepsy is one of the most common central nervous system (CNS) diseases, affecting over 50 million individuals globally [1]. Currently, anti-epileptic drugs are recognized as the major therapeutic strategy; however, these drugs have unsatisfactory effects on the therapy for epilepsy, and drug resistance represents a non-negligible problem [2]. Clinical investigation has confirmed that only 66% efficiency of anti-seizure drugs is achieved, even in high-income countries [3], and half of patients with epilepsy still present seizures after treatment with anti-epileptic drugs [4]. Therefore, it is essential to investigate additional prospective targets and strategies for epilepsy therapy through extensive research on the mechanism.

Generally, epilepsy is considered to be a process of synchronization and amplification mediated by an imbalance between excitatory and inhibitory synaptic transmission [5], and the abnormal connectivity of synapses in the brain contributes greatly to epileptic network formation and increases sensitivity to seizures [5, 6]. Astrocytes, the most abundant cells in the CNS, can not only support neurons but also regulate the stabilization and plasticity of synapses [7]. Christopherson et al. reported that co-culture with mature astrocytes is essential for synaptogenesis of retinal ganglion cells (RGCs). The results suggest that astrocytes secrete vital extracellular signaling molecules for synapse formation. Further analysis confirmed that the macromolecular protein secreted by astrocytes is thrombospondin-1 (TSP-1) [8].

A member of the thrombospondin family, TSP-1 is a multidomain extracellular matrix protein [9, 10] that is expressed highly in the developing brain, exhibits low expression in the adult brain [11], and regulates neuronal migration, as well as synapse formation and growth [12, 13]. Previous studies have confirmed that increased secretion of TSP-1 from activated astrocytes contributes to synaptogenesis and angiogenesis, eventually promoting epilepsy development [14, 15]. A critical protein secreted by astrocytes, TSP-1 supports dendritic spine development, which plays a vital role in modulating synaptic and circuit alterations [16]. Previous studies have reported that the expression of astrocyte-secreted TSP-1 reaches a peak that is consistent with the synaptogenic period and the initiation of excitatory synaptogenesis in vivo [17]. The above work suggests that astrocytic TSP-1 has a vital effect on synaptogenesis. Interestingly, as an endogenous angiogenesis inhibitor, TSP-1 reportedly contributes to angiogenesis [18]. Furthermore, it has been confirmed that TSP-1/transforming growth factor (TGF-β1)/phosphorylated small mothers against decapentaplegic (pSmad2/3)/vascular endothelial growth factor (VEGF) pathway, which is regulated by P2 receptors (especially P2Y4), promotes angiogenesis in rats with kainic acid-induced status epilepticus [18]. The results above demonstrate that TSP-1 plays an important role in the formation of epileptic networks through the regulation of synaptogenesis and angiogenesis.

Besides the contributions to synaptogenesis and angiogenesis [18, 19], studies have suggested that TSP-1 is also involved in multiple complex processes, which are thought to be closely associated with epilepsy formation and development, including apoptosis [20], neuronal injury [21], neuroinflammation [22], oxidative stress [23], and receptor or ion channel dysfunction [24]. In this review, we summarize the demonstrated and potential contributions of TSP-1 to epilepsy development and provide a systematic and theoretical basis for future studies on the mechanisms underlying epilepsy development and the potential strategies through TSP-1 targeting.

Structure of TSP-1

TSP-1 was the first member of the TSP family to be reported. It is also the most extensively studied extracellular matrix protein [25]. The TSP family contains five proteins: TSP 1–5 [26]. TSP-1 was discovered by Baenziger et al. [27] in 1971. Lawder et al. [28] have revealed that intact TSP-1 protein is a homotrimer weighing 420 kDa and mature polypeptide chains of TSP-1 are 180 kDa [25]. Since its discovery, TSP-1 has been defined as a glycoprotein associated with the α-granules during platelet activation [29]. TSP-1 has a complex multidomain structure, which is considered the basis of interactions with various molecules. TSP-1 comprises trimer subunits with disulfide bonds and each subunit includes 1152 amino-acid residues [30], which are linked by disulfide bonds between cysteines 252 and 256 [25]. Each subunit of the TSP-1 trimer comprises β-sandwich domains made up of the amino-terminal (N-terminal) and carboxyl-terminal (C-terminal) connected with a flexible and long arm [30, 31]. The arm region includes two cysteine residues (participating in the formation of the trimer), a procollagen homologized region, and three types of repeated domains—types I, II, and III [32]. The different domains of the TSP-1 protein exhibit different characteristics and functions under various conditions. For example, the N-terminal might interact with heparin, heparan sulfate proteoglycan, and low-density lipoprotein receptor-related protein 1 (LRP 1) [33]. The type III repeats of TSP-1 contain numerous aspartic acid residues that form one or two sequences of each repeat, similar to Ca²⁺-binding motifs, forming a structural foundation for binding with Ca²⁺. This feature is vital for the functional properties of TSP-1 [34]. It is clear that the multiple functions of TSP-1 may mainly depend on the interactions of its various structural domains with cellular molecules.

TSP-1-Interacting Molecules

The structural characteristics of TSP-1 provide reliable action sites for multiple types of molecules. Although the complete specific networks of the TSP-1 interaction remain unclear, various TSP-1-interacting molecules have been successively identified. Interestingly, studies have shown that the multifarious functions of TSP-1 depend on the molecules that interact with it [35, 36], including cytokines, growth factors, and structural components of cell receptors [37] (Fig. 1). Moreover, bioinformatics analyses have shown that TSP-1-mediated signaling affects numerous processes, such as neural development, angiogenesis, cell proliferation, adhesion, and motility [38]. Therefore, it is necessary to understand the contributions of TSP-1 in the epileptic network by appreciating of multiple functions of molecules interacting with TSP-1. Some major molecules interacting with TSP-1 are listed as follows.

Fig. 1.

The simple structure and interacting molecules of TSP-1.

CD47

CD47 is a highly hydrophobic glycoprotein, which is composed of an IgV-like extracellular domain, a five-span transmembrane region, and a short intracytoplasmic tail lacking a signaling motif [39]. Lehrman et al. [40] found that CD47 colocalizes with the pre- and postsynaptic markers, which indicated that CD47 might be localized in synapses. Previous work has reported that the binding sequences of CD47 are identified by the C-terminal of TSP-1 [41]; however, how TSP-1/CD47 signaling regulates synaptogenesis and epileptogenesis in the brain remains unclear. Nonetheless, the interactions of TSP-1 and CD47 regulate multiple functions like cellular adhesion, apoptosis, migration, and neuroimmunity, which are believed to be related closely to neurodegenerative disorders [42]. In addition, the TSP-1/CD47 interaction reportedly plays a key role in the modulation of cell invasion and angiogenesis in glioblastoma [43]. Such findings suggest that TSP-1/CD47 might be associated with the related pathological progress of epileptic networks.

CD36

CD36 is considered to possess a CLESH-1 homology motif that interacts with the type I repeats of TSP-1 [44]. Similar to CD47, CD36 is identified as a receptor for TSP-1 and is vital for inflammation and angiogenesis. A previous study has demonstrated that TSP-1/CD36 exerts anti-tumor activities by inhibiting angiogenesis in the tumor microenvironment [45]. Conversely, the evidence demonstrated that TSP-1 can increase VEGF receptor 2 levels by binding CD36 [46]. The Ortiz-Masià research group has reported that hypoxia induces the phagocytosis of macrophages significantly, increasing the expression of TSP-1 and CD36, and TSP-1/CD36 may contribute to this inflammatory process [47]. Besides, CD36/TSP-1 is reported to participate in the induction of oxidative stress [48].

TGF-β1

TGF-β1 is a regulatory protein that is activated by TSP-1 [49] and participates in angiogenesis, scar deposition, inflammation, astrocytic phenotype, and mobility regulation [50]. A recent study reported that TSP-1 plays a major role in the molecular mechanism of synaptogenesis, and the progression has been demonstrated to be connected with the interaction between TSP-1 and neuroligin 1/TGF-β1 [51]. Besides, TSP-1/TGF-β1/Smad signaling pathways can influence angiogenesis via diversely regulating the expression of VEGF [52]. These processes are closely related to epileptic network formation.

Lipoprotein Receptor-Related Protein-1 (LRP1)

TSP-1 reportedly interacts with LRP-1 [53]. The interaction could promote VEGF clearance, inhibiting angiogenesis. Notably, it has been reported that activated astrocytes secrete TSP-1, which, upon binding to LRP1 in the synapse, promotes synaptic recovery [54]. In addition, TSP-1 has been shown to be associated with LRP-1 in T cells. T cell motility is triggered through the combination of TSP-1 with LRP1 and the co-related receptor calreticulin [55]. Such evidence indicates that the interactions between LRP-1 and TSP-1 regulate the function of synapses, angiogenesis, and immunity, and therefore might be an underlying contributor to epileptogenesis.

α2δ-1

As a voltage-gated Ca²⁺ channel receptor, α2δ-1 is considered to be a key target of the commonly prescribed anti-epileptic drug Gabapentin [56]. Gabapentin is thought to block the binding of TSP-1 and α2δ-1, and in turn, inhibit the formation of synapses [57]. A previous study found that excitatory neurons (including RGCs) express high levels of α2δ-1, which localizes in synapses [17]. Overexpression of α2δ-1 increases excitatory synaptogenesis, whereas knockout leads to defects in synapse formation [17]. Hence, TSP-1-α2δ-1 is crucial for synapse formation, which participates in epileptogenesis.

Consequently, the interaction of TSP-1 with diverse proteins contributes to a wide spectrum of cellular functions. Therefore, it is beneficial to understand the mechanisms of epileptic network formation by focusing on these potential molecular interactions with TSP-1 in the brain.

Increased Expression of TSP-1 Promotes Synaptogenesis in Epileptic Network Formation

Astrocyte-Secreted TSP-1 Promotes Synaptogenesis

Studies have revealed that astrocytes, the most abundant glial cells in the brain, play an important role in synaptic connectivity, regulation of synaptic function, and provision of growth factors [24, 58]. Because astrocytes are necessary for synaptogenesis in co-cultured RGCs, Christopherson et al. [11] further analyzed the soluble macromolecular proteins secreted by astrocytes. Based on the relative molecular weight characteristics and a heparin-binding region, the active substance promoting synapse formation was demonstrated to be TSP-1. Further studies have also confirmed that TSP-1 purified from human platelets significantly increases the number of synapses in cultured RGCs even in the absence of astrocytes [8]. Similarly, animal experiments have confirmed that astrocyte-secreted TSP-1 contributes to synapse formation and is a vital factor in synaptogenesis [11]. A study on the distribution of TSP-1 in newborn mouse brains showed that on day 8 after birth, TSP-1 is distributed widely in the cortex, superior thalamus, and retina, whereas it almost disappears by day 21 [59]. Tagnaouti et al. [60] and Danjo et al. [61] revealed that the transcripts of TSP-1 increase significantly during mouse embryonic development, whereas TSP-1 expression is reduced significantly during the late period of development and early adulthood. These results suggest that elevated TSP-1 levels are highly consistent with the period when synaptic formation is highly active [59].

Furthermore, TSP-1 plays an important role in the regulation of nervous system function by promoting synapse formation [62]. TSP-1 knockout results in a significantly reduced number of synapses in the cortex of rats [10]. Moreover, in Alzheimer's disease and brain trauma, studies have found that synaptic formation and plasticity in TSP-1 knock-out mice are significantly inhibited, leading to abnormalities in learning, memory, and behavioral function [63, 64]. In human astrocytoma infected by Zika virus, studies have shown that the expression of TSP-1 is closely consistent with synapse formation and plasticity [65]. The investigation of neural development-related intellectual disability confirmed that TSP-1 expression in astrocytes of fragile X syndrome is reduced remarkably [66]. Similarly, decreased TSP-1 expression has been reported in Down’s syndrome astrocytes, accompanied by defects in synapse formation and neural function [66, 67].

However, how does TSP-1 promote synaptogenesis? Further experimental data have shown that the critical transcriptional factor, TGF-β1, is upregulated and activated, accompanied by increased TSP-1 levels, to promote the expression of synaptophysin in RGCs [68]. Although the synaptic number of mouse neurons, which were co-cultured with human astrocytes, increased by 260%, administration of a TGF-β1 pathway inhibitor completely prevented the increase of synaptogenesis. Moreover, compared with neuronal culture alone, TGF-β1 administration in the culture medium resulted in a significantly increased number of synapses, and the synaptic ultrastructure was similar to that of normal [69]. Such evidence suggests that TSP-1 plays a vital role in synaptogenesis via regulation of the downstream molecule, TGF-β1.

Besides, TSP-1 is involved in the repair and regeneration by inducing synaptogenesis [70]. Studies have reported that TSP-1 secreted by astrocytes promotes the synaptic regeneration of neurons and the growth of axons [10, 71]. In addition, TSP-1 accelerates synaptogenesis in young hippocampal neurons in the human brain [72]. Khakh et al. [73] reported that, in a hyperactivity model, medium spiny neurons (MSNs) of the striatum activate astrocyte-related signaling pathways. During this pathological progression, the level of TSP-1 secreted by astrocytes increases and is accompanied by enhanced excitatory synaptic connectivity and transmission. All these changes are reversed by inhibiting TSP-1. Such studies reveal the key role of astrocyte-secreted TSP-1 in synapse formation.

Therefore, the in-depth study of the function and mechanism of TSP-1 in synapse formation is of great importance for understanding the functional regulation of the CNS and the contribution of TSP-1 to epileptic network formation.

Regulation of TSP-1 Secretion

A vital protein that is mainly secreted by astrocytes, TSP-1 secretion is considered to be regulated by various factors, including cytokines, extracellular matrix components, and intracellular signaling pathways [74]. For instance, researchers have found that some extracellular matrix components like adenosine triphosphate (ATP) or collagen promote TSP-1 secretion [75, 76]. Meanwhile, Hisaoka-Nakashima et al. [77] reported that lysophosphatidic acid activation stimulates TSP-1 secretion via the extracellular signal-regulated kinase (ERK), MAPK, and JNK signaling networks in astrocytes. Conversely, it has been reported that activation of the phosphatidylinositol 3 kinase (PI3K)/Akt/mTOR singling pathway downregulates the TSP-1 secretion in human tumor-derived endothelial cells [78]. In addition, some transcription factors, such as NF-κB and AP-1, have been demonstrated to influence TSP-1 secretion via the regulation of genetic transcription under different conditions [79].

Previous studies have revealed multiple mechanisms that regulate TSP-1 secretion from astrocytes. In general, this regulation is divided broadly into two types: intracellular and extracellular [80, 81]. Some intracellular signaling pathways in astrocytes, such as Wnt and K-ras, have been reported to repress the secretion of TSP-1 in colonic tumorigenesis [82]. Therefore, stimulation of the expression of TSP-1 is considered a potential therapeutic strategy. Moreover, cytokines (such as TNF-α) have been reported to induce the synthesis and secretion of TSP-1 via the Akt and P38 MAPK signaling pathways [83]. Conversely, astrocytes can regulate the secretion of TSP-1 through extracellular signals [14]. Specifically, when astrocytes are stimulated by external factors, extracellular receptors are activated, which in turn prompt astrocytes to secrete TSP-1 [84]. This secretion process can be regulated through various signaling pathways, including ERK [85] and PI3K [78].

In addition, the extracellular receptor P2Y4 is a specific receptor that is located on the membrane of astrocytes [86]. Recently, it has been confirmed that activation of the P2Y4 receptor increases intracellular Ca²⁺ [87]; furthermore, increased astrocytic Ca²⁺ signaling can promote TSP-1 release [88]. Therefore, it is reasonable to speculate that P2Y4 receptor and astrocytic Ca²⁺ signaling are involved in TSP-1 secretion. In addition, the activation of the P2Y4 receptor can affect TSP-1 synthesis and secretion by regulating the expression of transcriptional factors and cytokines within cells [14, 89]. For example, Hu et al. [90] reported that TSP-1 is upregulated significantly in activated astrocytes in the striatum of mice, accompanied by increased synaptic inputs from upstream neurons and the growth of MSNs, causing behavioral hyperactivity. Furthermore, the increased TSP-1 secretion is regulated by the purinergic P2-type receptor [91], especially P2Y4, which are vital regulator of TSP-1 secretion in activated astrocytes [14, 69]. In addition, Koizumi et al. [60] reported that, in a neurological pain model, the expression of metabotropic glutamate receptor 5 (mGluR5) in somatosensory cortex astrocytes promotes TSP-1 secretion by increasing Ca²⁺ activity, eventually leading to excessive synapse formation and incorrect connectivity of the pain network. Xiao et al. [92] also revealed that hyper-glucose conditions lead to astrocyte dysfunction and reduce TSP-1 secretion, which might be associated with the activation of toll-like receptor 9 signaling.

The Possible Mechanism of TSP-1 in Synaptogenesis

TSP-1 reportedly triggers a series of signal transduction pathways by binding related molecules on the surface of neurons, thus promoting the formation and development of synapses. Among them, TSP-1 can bind with CD47 on the surface of neurons and activate CD47-related signaling pathways (such as CD47-SIRRP α), which promotes synapse formation [93]. In addition, TSP-1 can bind to other molecules on the surfaces of neurons, such as αvβ3 integrins and LRP1, which have been confirmed to influence synapse formation by regulating extracellular matrix assembly and intracellular signaling [94, 95]. Studies have also shown that astrocyte-derived TSP-1 can regulate the assembly and breakdown of extracellular matrix around neurons, thereby affecting synapse formation and development [96]. Specifically, TSP-1 can increase adhesion and interaction between presynaptic and postsynaptic neurons, besides promoting the assembly of extracellular matrix, thus promoting excitatory synapse formation and development [65]. In addition, TSP-1 can affect synapse formation and development by regulating extracellular matrix molecular activity: TSP-1 can reportedly inhibit the activity of Glypican-4, thereby promoting the formation and development of excitatory synapses [60].

TSP-1 binding to the Leu-Ser-Lys-Leu (LSKL) peptide sequence in the TGF-β1 complex leads to TGF-β1 activation, and preventing TSP-1 binding by administration of exogenous LSKL impedes TGF-β1 activation [70]. Our previous results also confirm that an increase in the number of synapses in the brains of amygdala-kindled seizure rats accompanies elevated levels of TSP-1 and TGF-β1. Similar to the roles of intervention in TSP-1 expression, restraining the TGF-β1 activation by administering exogenous LSKL significantly attenuated synaptogenesis and epileptic development [14]. Such results suggest that the TSP-1/TGF-β1 pathway is a critical promoter of synaptogenesis and epileptogenesis [14] (Fig. 2). Further investigations are required to uncover the precise mechanisms underlying the role of TSP-1 in synapse formation.

Fig. 2.

Regulation of TSP-1 secreted by activated astrocytes and potential mechanism of TSP-1 in excitatory synaptic formation.

Synaptogenesis is the Basis of Epileptic Network Formation

The occurrence and development of epilepsy involve the progression of abnormal neural network formation based on synaptogenesis [97]. Numerous clinical and animal studies have confirmed that some complex and intractable epilepsies originate from abnormal neuronal protrusions in the temporal lobe region [98]. Evidence shows that neurons are highly connected through excitatory synapses to overcome the feedback inhibition of interneurons and eventually form epileptic networks [8]. As a key participant in neuronal activity, abnormal excitatory synaptogenesis is critical in building hyperexcitatory neural circuits that contribute to epilepsy [99, 100]. Recent studies have indicated that the transmission regions of epileptic signals in the brain are strongly correlated with the number of synaptic changes [14]. In addition, there is evidence that net excitation or inhibition in the new neural circuits depends on the number of synapses formed on principal cells or interneurons [101]. Although the two types of synapses are found in normal and epileptic rats, the newly formed synapses sprouting in the inner molecular layer of the dentate gyrus (DG) are mainly on the principal cells and contribute to the recurrent excitatory circuitry [101, 102]. It has been reported that mossy fiber sprouting plays a critical role in the reorganization of circuits during seizures [103]. During epileptic seizures, the surviving mossy cells participate in the formation of an aberrant synaptic network, establishing excessive positive feedback in the granule cells of the DG and CA3 in the hippocampus, which are considered pivotal for promoting the connectivity of excitatory synapses [104]. In addition, Freiman et al. [105] found that sprouted mossy fibers not only lead to excitatory synaptic connectivity but also produce extra neural innervation on preserved inhibitory interneurons, eventually causing an imbalance between excitation and inhibition in the brain.

Therefore, synaptogenesis, especially excitatory synaptogenesis, plays a key role in epileptic network formation and is a critical factor in inducing epileptogenesis [106]. In addition, TSP-1 has been verified as a vital factor in excitatory synapse formation [107]. Consequently, targeting TSP-1 is beneficial for inhibiting epileptic network formation through the intervention in synaptogenesis [14].

The Upregulation of TSP-1 can Contribute to Epileptogenesis by Promoting Angiogenesis

Contribution of TSP-1 to Angiogenesis Regulation

Physiologically, the angiogenic progress is maintained by the balance of the angiogenic promotors and inhibitors [108]. TSP-1 is considered an endogenous angiogenesis inhibitor for repair when the tissue is injured under physiological conditions [109]. The anti-angiogenic function of TSP-1 can be mediated by its structural three type I repeats [110]. However, when the pro- or anti-angiogenic factors are dysregulated under pathological conditions, it is believed to favor angiogenesis [98]. TSP-1 is thought to be a vital factor regulating angiogenic progress under a pathological internal environment. Research has demonstrated that TSP-1 promotes angiogenesis through binding with CD47 (integrin-associated protein), in turn enhancing the repair of post-ischemic damage [111]. The extracellular matrix is a key part of the angiogenesis process, supporting cell migration and vascularization [112–114]. In patients with gliomas, the increased TSP-1 levels, which are mainly distributed in intracellular spaces or the cytoplasm, are closely related to the process of angiogenesis in the gliomas [115]. In the human prostate cancer cell line, dynamic changes in TSP-1 expression level have been confirmed during angiogenesis, and its expression level is positively correlated with the degree of angiogenesis [115, 116]. A previous study confirmed that TSP-1 promotes vascular proliferation by activating α9β1 integrin in a quail chorioallantoic membrane system experiment. Inhibition of α9β1 integrin reduces the TSP-1-mediated proangiogenic activity in vivo [117], which indicates that TSP-1/α9β1 integrin signaling is important for angiogenesis. Yang et al. [118] found that thrombin participates in angiogenesis in rats with cerebral hemorrhage by increasing TSP-1/2 expression. After administering hirudin, a thrombin inhibitor, the levels of TSP-1/2 are reduced, and angiogenesis is inhibited. In addition, in an ischemic stroke model, upregulated miR-487b promotes neovascularization by directly targeting TSP-1. Further analysis has suggested that increased TSP-1 expression is due to miR-487b binding to TSP-1 mRNA. The results indicate that miR-487b may mediate angiogenesis in ischemic stroke by increasing TSP-1 levels [119].

Previous studies have shown that the activation of Smad 2/3 mediated by TGF-β1 can induce angiogenesis by accelerating the expression of angiogenic factors, such as VEGF [120]. Furthermore, it has been confirmed that TGF-β1 is activated by TSP-1 [121] and that the N-terminal domain of TSP-1 is responsible for angiogenesis [122]. Moreover, TSP-1 secretion is mainly regulated by the P2Rs receptor (mainly the P2Y4 receptor) in astrocytes [15]. In rats with KA-induced status epilepticus, synchronously elevated levels of TSP-1, TGF-β1, and phosphorylated Samd 2/3 are accompanied by angiogenesis. Angiogenesis is significantly attenuated by blocking P2Y4, reducing TSP-1 expression, or TGF-β1 activation. This strongly indicates that the P2Y4/TSP-1/TGF-β1/pSmad2/3 pathway is a pivotal component of angiogenesis in epilepsy [18].

In addition, TSP-1 affects angiogenesis by regulating the expression and activity of the pro-angiogenic factors, which are believed to be essential for angiogenesis [116]. Some cytokines have been reported to be involved in promoting angiogenesis, like VEGF and basic fibroblast growth factor [116]. For instance, NF-κB is considered an important transcription factor that induces the expression of the VEGF gene [123], and TSP-1 increases VEGF gene transcription by activating the transcription factor NF-κB [124]. Ghorbanzadeh et al. [125] reported that the TSP-1/NF-κB/VEGF signaling pathway participates in promoting angiogenesis in the heart of aged rats.

In summary, TSP-1 plays an important role in angiogenesis regulation. It is involved in the regulation of various links of angiogenesis by regulating the proliferation and migration of endothelial cells, inducing their apoptosis, regulating their differentiation and function, and regulating the expression and activity of angiogenesis-related factors [115, 125, 126]. The in-depth studies of the role of TSP-1 in the regulation of angiogenesis help to reveal the molecular mechanisms of angiogenesis in related diseases.

Angiogenesis is a Crucial Participant in Epileptogenesis

Angiogenesis, an important event in many diseases and first described by Spielmeyer in 1925, is a key factor involved in the development of epilepsy [127]. Accumulating evidence since 2007 suggests that intractable temporal lobe epilepsy (TLE) is associated closely with angiogenesis [128, 129]. Numerous genes are responsible for the VEGF signaling pathway in patients with TLE, which is associated with GABA or glutamate synaptic transmission [130]. Clinical studies have also reported that vascularization and increased permeability of the blood-brain barrier (BBB), leads to large leakage of serum proteins and inflammation in the hippocampus of patients with TLE [128, 131]. The BBB depends on intercellular tight junctions and adherence [132]. The study demonstrated that, in epilepsy, angiogenesis may lead to BBB permeability; furthermore, BBB leakage is associated with structural aberrations during epileptic seizure and activates the glutamate signaling pathway via A2 cytosolic phospholipase [133]. In addition, BBB leakage causes several serum proteins, such as albumin, to be released into the parenchyma, which can lead to epileptic focus production [134, 135]. In KA-induced status epilepticus, reduced TSP-1 expression results in attenuated angiogenesis, vascular leakage, and inhibition of epileptogenesis [18].

Angiogenesis is a key pathological process that contributes to epileptogenesis, and TSP-1 has been confirmed to be an active participant in angiogenesis. Moreover, previous studies have confirmed that TSP-1 participates in epileptogenesis via angiogenesis.

TSP-1 Participates in Epilepsy by Regulating the Level of Oxidative Stress

TSP-1 Exerts a Crucial Role in Oxidative Stress

Astrocytes can respond to changes in oxidative stress levels in the brain and play diverse roles in regulating oxidative stress [136]. Besides their roles in the decomposition and clearance of free radicals, astrocytes can also be the main source of detrimental reactive oxygen species (ROS), leading to neuronal injury [137]. TSP-1 also promotes ROS generation [138]. The expression of TSP-1 is upregulated in many oxidative stress-related diseases, such as ischemia-reperfusion injury and atherosclerosis [139]. For example, synchronously increased TSP-1 levels and oxidative stress have been detected within an hour of ischemia [137]. Recently, Thom et al. [140] described a pre-feedback inflammatory/oxidative stress cycle mediated by TSP-1. They reported that in brain CO poisoning, astrocytes induce TSP-1 expression; subsequently, TSP-1 is released into the blood and promotes CD36 (a ligand of TSP-1) expression and oxidative stress. Such studies indicate that increasing TSP-1 levels could be an important contributor to oxidative stress.

However, previous studies have demonstrated that TSP-1 may have a neuroprotective effect on Aβ-treated mouse hippocampal neuroblastoma cells. Further results have shown that TSP-1 inhibits the transmission of Drp-1 into mitochondria and mediates mitochondrial fission by maintaining phosphorylated-Drp1 levels and the stability of mitochondrial function [141]. Mitochondrial dysfunction is one of the major processes involved in oxidative stress [142]. The above studies provide novel insights into the relationship between TSP-1 and oxidative stress. There are two contrasting roles of TSP-1 in oxidative stress regulation, and its specific roles during epileptogenesis remain to be explored.

Oxidative Stress Level is Critical in Epilepsy Formation and Development

The brain is sensitive to oxidative stress due to hyperoxic metabolism. Overproduction of free radicals has been confirmed to lead to neuronal hyperexcitability and injury, which participate in epilepsy initiation and progression [143]. Mitochondria are the main sites of ROS production during various biochemical processes, including the tricarboxylic acid cycle [144]. Overproduction of ROS can damage mitochondrial function, which, in turn, affects the synthesis of many enzyme complexes in the electron chain [145] and leads to mitochondrial disorders [146]. This pathological progression is conducive to epilepsy formation and development [146]. Multiple animal models of epilepsy, such as pilocarpine and kainic acid, have confirmed mitochondrial dysfunction and elevated levels of oxidative stress [147]. Moreover, excessive ROS-induced mitochondrial injury leads to energy metabolism disorders, which play a vital role in the occurrence and development of epilepsy [148]. Oxidative stress induced by excessive ROS production may eventually lead to the onset of epilepsy [149]. Conversely, decreasing ROS levels reduce the release of excitatory amino-acids and attenuate epileptic seizures [148, 150]

In summary, TSP-1 is secreted mainly by activated astrocytes and plays a vital role in oxidative stress in the brain. Based on the close relationship between oxidative stress and epilepsy, TSP-1 may play a pivotal role in epilepsy development by oxidative stress.

TSP-1 Might Participate in the Progress of Epilepsy by Mediating Neuroinflammation

A previous study detected transiently increased levels of TSP-1 in inflamed tissues [151]. CD47, a natural ligand of TSP-1, is a homologous trimeric extracellular matrix protein that mediates neuroinflammatory pathways [152, 153]. Studies have reported that the TSP-1/CD47 pathway plays a major role in numerous neurological disorders, such as decompression sickness [154], glioblastoma [43], spinal cord injury [155], stroke [42], Alzheimer’s disease [156, 157], and neonatal meningitis [151]. For example, in the Escherichia coli K1-induced-neonatal meningitis model, upregulation of TSP-1 and CD47 is accompanied by inhibition of dendritic cell maturation. Subsequently, blocking TSP-1 or CD47 protects the brains of newborn mice against neuroinflammation and damage caused by E. coli K1 [151]. In addition, CD36 is a key molecule that interacts with TSP-1 in neuroinflammation [158]. A previous study demonstrated that the activation of phosphatidylserine/CD36/TGF-β signaling significantly counters anti-inflammatory effects on the neuroinflammation-induced injury of rats [159]. Although the specific molecular mechanism is still unclear, it is hypothesized that TSP-1 may regulate neuroinflammation by BBB breakdown [160]. In addition, previous research has revealed that TSP-1 can result in the leakage of the BBB, causing the neuroinflammatory response and upregulation of the levels of some chemokines like macrophage inflammatory protein 2. This progress is regulated by Lysophosphatidic acid receptor 1 (LPA1) via the ERK, MAPK, and JNK signaling network [161].

Epileptic seizures are closely associated with elevated and persistent inflammatory reactions in the brain [162]. Inflammatory reactions are induced by the release of inflammatory agents like IL-1β, which can activate NMDA receptors [163]. Neuroinflammation-induced BBB damage is believed to be a crucial factor in epilepsy [164]. Conversely, systemic or focal inflammation would lead to aberrant neural connectivity and over-excitatory network formation, eventually causing epileptogenesis [165, 166]. Therefore, it is reasonable to assume that TSP-1-mediated neuroinflammation may be involved in epilepsy development. The effects of TSP-1 on neuroinflammation during epileptogenesis require further confirmation.

TSP-1 Might Play a Pivotal Role in Epilepsy by Regulating Neuronal Injury

Previous studies have demonstrated that TSP-1 expression is generally low in mature brains under physiological conditions [167]. However, significantly increased TSP-1 levels have been detected during neuronal injury, indicating that TSP-1 might be involved in neural damage [168]. As a vital member of the matricellular family, TSP-1 regulates apoptosis under numerous physiological and pathological conditions [169]. Previous studies have reported that TSP-1 is expressed mainly in activated or stressed astrocytes and endothelial cells to induce apoptosis and is believed to be one of the most critical factors causing injury [170–172]. When neurons begin to die, TSP-1 secretion is increased synchronously due to ATP release [173], and the TSP-1-CD47 pathway builds a bridge between cell phagocytosis and death [174]. This leads to a peak in microglial proliferation and phagocytosis due to induction by damaged neurons [166]. Lo et al. [175] reported that TSP-1 binding to CD47 mediates cell death through caspase-3-dependent or -independent pathways. Secondly, the binding of TSP-1 and CD47 can inhibit cellular regeneration and migration [176]. Furthermore, TSP-1 can affect the growth and connection of neurons by regulating the composition and structure of the extracellular matrix [177]. Recently, Norenberg et al. [178] found that astrocytes are dysfunctional and lose their neurotrophic function during neuronal injury, which may be associated with the TSP-1-TDP-43 signaling pathway.

Neuronal injury is a critical feature of epileptogenesis. Increased levels of excitatory neurotransmitters, including glutamate and acetylcholine, are critical factors in the pathophysiology of neural injury [179]. Neuronal injury promotes epileptogenesis [21]. In addition, astrocytes are thought to partly maintain the balance between excitation and inhibition by absorbing excessive glutamate during seizures. However, neural injury hampers this progress due to elevated synaptic release and disordered glutamate uptake [180]. Therefore, TSP-1 may participate in epilepsy formation and development by regulating neuronal injury.

TSP-1 Probably Contributes to Epileptogenesis by Interacting with Ca²⁺ and the Ca²⁺ Voltage-Gated Channel Receptor α2δ-1

A previous study has reported that increased dendritic release of Ca²⁺ plays a key role in synchronous neuronal firing induced by crosstalk between neurons and astrocytes [181]. Especially astrocytic Ca²⁺ is involved in many supportive roles in regulating synaptic transmission. Moreover, substantial evidence has demonstrated that Ca²⁺ is an active participant in the release of glutamate from active astrocytes [130, 131], and is a promotive contributor to epilepsy.

TSP-1 is an important regulator of Ca²⁺ homeostasis as the C-terminal signature domains form a C-terminal β-sandwich, which carries many Ca²⁺ binding sites, forming coordination bonds with it, thus changing the conformation and function [34, 182]. In addition, TSP-1 can maximally combine with 30 Ca²⁺ ions at a time [34, 117], and experimental data have revealed that TSP-1 participates in regulating the influx and concentration of Ca²⁺ in cells [183, 184]. For example, Martin-Manso et al. [183] have found an elevated level of intracellular Ca²⁺ after the addition of TSP-1. Besides, research has also indicated that TSP-1 treatment may lead to an influx of Ca²⁺ in human red blood cells [184].

In addition, TSP-1 regulates synapse formation and plasticity by interacting with Ca²⁺ channel receptor α2δ-1 [185]. The α2δ-1 subunit, which is mainly located at neurons [186], is considered pivotal for the formation and maturation of excitatory synapses in the cortex and plays an important role in the trafficking and kinetics of the Ca²⁺ voltage-gated channel [187, 188]. For instance, Kim et al. [189] have reported that elevated levels of Ca²⁺ promote the release of TSP-1, which induces excitatory synaptogenesis via binding with the α2δ-1 receptor. Besides, excitatory synapse formation is controlled by interactions with TSP-1 and the α2δ-1 receptor via Rac1, which suggests the potential mechanisms of both the physiology and pathology of synaptic development [190, 191]. These mechanisms help maintain the steady-state Ca²⁺ and Ca²⁺ voltage-gated channel within neurons to prevent excessive Ca²⁺ accumulation from causing damage to neurons [189, 190]. TSP-1 regulates excitatory synaptic formation by binding with α2δ-1 of the Ca²⁺ channel in the intracerebral hemorrhage [188] and kindling models [5].

Previous studies have demonstrated that the interaction of TSP-1 and Ca²⁺ or the Ca²⁺ voltage-gated channel plays numerous roles in the nervous system; it is involved in promoting neurodevelopment and synaptic formation, neuroprotection and repair, and regulation of neuroinflammation and immune responses, among other processes. Such molecular progresses are believed to be critical factors for the formation of epileptic networks. Although the specific mechanisms of the interactions of TSP-1 and Ca²⁺ in regulating epilepsy formation are still unclear, TSP-1 is causally linked to Ca²⁺ [59, 187], and targeting TSP-1 is a prospective epileptic therapy.

Conclusions

In summary, although epilepsy is one of the most complex medical challenges today, the specific mechanisms of the neuronal disease remain unclear. Therefore, exploring the pathogenesis of epilepsy is vital for the development of therapeutic targets and strategies.

TSP-1 is a prospective protein discovered in recent years that contributes significantly to epileptic network formation. Studies have suggested that TSP-1 may contribute to epilepsy formation and development in multiple ways, such as by regulating synaptogenesis, angiogenesis, neuroinflammation, and Ca²⁺ homeostasis (Fig. 3). Based on the potentially vital role of TSP-1 in epileptogenesis, targeting TSP-1 may be a prospective strategy for treating epileptogenesis and epileptic seizures.

Fig. 3.

Other factors underlying the regulation of TSP-1 in epileptogenesis, including angiogenesis, neuronal injury, oxidative stress, neuroinflammation, and Ca²⁺ homeostasis.

Conflict of interest

The authors declare that they have no conflicts of interest.