Neuroinflammation (RHOB, TLR1, TNFRSF1A, LYN, CASP1, PRKCD, FCGR2A, IFNGR1)
Neuroinflammation is known to promote temporal lobe epileptogenesis and epileptogenicity [
21,
22]. In TLE, chronically activated systemic lymphocytes express inflammatory cytokines and traffic within the brain parenchyma exacerbating inflammation and neurotoxicity [
8,
23,
24] (Fig.
3). Several leukocyte DEGs identified in this study are involved in neuroinflammation and their upregulation was identified when comparing low to high seizure frequency.
The small GTPase, RHOB (Ras Homolog Family Member B; Rho-Related GTP-Binding Protein RhoB), activates the dual transcription factors, NF-kB and STAT3, which are involved in (a) acute inflammation and (b) neural differentiation and immune response with a positive correlation with seizure frequency, respectively [
25‐
27]. Specifically, among the classic RHO isoforms (RHOA, RHOB, RHOC), RHOB is uniquely endosomal and activates NF-kB, an acute inflammation transcription factor which is over-expressed in hippocampal CA1 and CA3 pyramidal neurons, reactive astrocytes and dentate granule cells and astrocytic processes in patients with medial temporal lobe epilepsy [
28‐
30]. In glioblastoma cell lines, RHOB knockdown has been shown to cause decreased cytokine-induced STAT3 activation and impaired STAT3 activity [
28]. Inflammatory pathways, including those involving STAT3, have been implicated in preclinical studies in the modulation and genesis of epilepsy after brain injury [
31]. The JAK/STAT pathway specifically is activated in the rodent pilocarpine model of status epilepticus (SE), which produces temporal lobe epilepsy and the inhibition of STAT3-regulated gene transcription is shown to decrease long-term spontaneous seizure frequency following onset of status epilepticus [
32]. In human epilepsy, serum levels of all STATs are elevated, with STAT3 being the most profound at a nine-fold increase above baseline [
21].
Another gene identified in our study, PRKCD (PKCδ, Protein Kinase C Delta), is involved in the epilepsy inflammatory response and has been shown to increase neuronal excitability and epileptogenesis [
33]. PKCδ is a proinflammatory, oxidative stress-inducing and epileptogenic factor in temporal lobe epilepsy and various isoforms of Protein Kinase C (PKC) are involved in epileptogenesis [
34]. PKCδ is involved in the inflammatory response to epilepsy which is known to increase neuronal excitability, decrease the seizure threshold, enhance blood–brain barrier permeability and produce epiletognenesis [
33]. PKCδ also activates several programmed cell death signaling pathways and hippocampal excitability in status epilepticus in rats [
35]. PKCδ is the immediate downstream target of Fyn, a non-receptor Src family of tyrosine kinase (SFK) [
36]. Hippocampal Fyn and PKCδ are increased following induction of the kainate model of status epilepticus and both Fyn and PKCδ demonstrate increased hippocampal microglial staining in epileptogenesis [
36]. Similarly, in a transgenic model of murine temporal lobe epilepsy, knockdown of microglia PKCδ eliminates the microglial proinflammatory inflammogen-induced response and decreases release of proinflammatory mediators, TNF-α, IL-1β, IL-6, and IL-12, reducing electrographic non- convulsive seizure frequency, epileptiform spiking and neuronal degeneration [
36].
Toll-like receptor 1 (TLR1) was also shown to be predictive of seizure frequency in our study and is implicated in temporal lobe epilepsy as a key transduction receptor of neuro- inflammation-induced epileptogenesis through glial production of inflammatory cytokines, IL-1β and TNF-α [
37]. TLRs represent a 10-member family of transmembrane proteins which detect damage associated molecular patterns (DAMPs) and have been implicated as key signal transducers in neuro-degenerative disorders and neuroinflammation-induced epileptogenesis [
38,
39]. Toll ligands promote neuronal and glial production of inflammatory cytokines including TNF-α, IL-1β
, IL- 6 and other inflammatory mediators of epileptogenesis [
38,
40,
41]. In patients with epilepsy, inflammation in brain tissue is predominately induced by the Toll-like receptors [
37]. Also of note, epileptogenic tissue shows upregulated TLR1in neurons, microglia and astrocytes which mediates both adaptive and innate immune responses [
37,
40]. Within this same pathway another gene detected in our study, the protein tyrosine kinase LYN (LYN Proto- Oncogene, Src Family Tyrosine Kinase), is known to activate toll-like receptors and regulates NF-kB while promoting over-expression of pro-inflammatory cytokines, IL-1β and TNF-α, accentuating neuronal hyperexcitability [
42,
43]. Importantly, LYN is a known regulator of neuroinflammation, neuronal excitability, and epileptogenicity and is a member of the non- receptor Src protein tyrosine kinase (SFK) family [
42]. SFKs are key signaling components of the immune response and microglial function and have been implicated in epileptogenesis [
36,
42,
44]. During epileptogenesis, SFK upregulation in hippocampal microglia occurs concurrently with upregulation of pro-inflammatory cytokines, electrographic non-convulsive seizures and increased epileptiform spiking [
36]. In contrast, in vitro inhibition of microglial LYN produces anti-inflammatory effects, including attenuated TNFα and IL-6 secretion [
43]. Additionally, SFK inhibition decreases in vitro hippocampal epileptiform discharge frequency and in vivo duration and seizure numbers in mice [
36,
45]. This correlation was also shown in our data.
Yet another gene detected in our study and known to be upregulated in human TLE is CASP1 (Caspase 1, Apoptosis-Related Cysteine Protease, Interleukin-1β Convertase). CASP1 is pro-inflammatory and pro-convulsant in human TLE and processes the major pro-inflammatory cytokine, Interleukin-1β (IL-1β), to an active secreted form, through leucine-rich repeat (LRR)- containing proteins (NLR) family member (NLRP) inflammasomes [
46‐
48]. In temporal lobe epilepsy (TLE), inflammasomes are involved in the seizure-induced degenerative process in both animals and humans [
49]. The NLRP1 inflammasome, expressed in both neurons and glial cells, exerts a crucial role in seizure-induced neuronal damage [
50‐
52]. Inflammasomes activate CASP1 and the transcription factor, NFkB, which both activate the pro-inflammatory cytokine, IL-1β, promoting epileptogenicity [
53,
54]. In human temporal lobe epilepsy patients, Caspase 1 is often upregulated in the hippocampi of patients with temporal lobe epilepsy and silencing of Caspase 1 or NLRP1 produces neuroprotective and antiepileptic effects which is also consistent with our data [
49]. Similarly, FCGR2A (FcγRIIA, Fc Fragment of IgG Receptor IIa) can modulate pro- and anti-inflammatory signaling via receptors of the Fc (fragment crystallizable) region of immunoglobulins (FcRs) which bridge the cellular and humoral pathways of the immune system [
55‐
57]. Specifically, FcγRIIA and FcγR mediate pro- inflammatory signaling pathways, neuro- and excitotoxicity, and lipid peroxidation which in turn promote epileptogenicity [
55,
56,
58‐
60]. Interestingly, cultured cortical and hippocampal cells, exposed to IgG-IC, induce FcγR-mediated IgG internalization, Erk phosphorylation and increased intracellular calcium [
58]. FcγR signaling is also responsive to the pro-inflammatory cytokine, IFNγ, and neuronal FcγRs contribute to kainic-acid brain neurotoxicity [
58].
Lastly, the gene IFNGR1 (Interferon Gamma Receptor 1, IFN-γ R1), which is pro- convulsant through activation of TNFR1 and TLR1 inflammatory pathways, operates through the pro-inflammatory cytokine, interferon- γ (IFN- γ). IFN- γ is produced by T and natural killer (NK) cells and microglia and binds to the IFN-γ receptor consisting of IFN-γR1 and IFN-γR2 subunits [
57,
61]. IFN-γR1 is expressed in both glial cells and neurons [
62]. Th1 cells secrete IFN-γ inducing microglia into a pro-inflammatory, cytotoxic M1 phenotype [
63]. In patients with temporal lobe epilepsy, peripheral lymphocytes are in a chronic state of activation and demonstrate increased IFN-γ expression compared to healthy controls [
24]. In patients with epilepsy, post-ictal and interictal peripheral blood concentrations of IFN-γ are also elevated, relative to healthy controls, and interictal IFN-γ concentration is positively correlated with seizure frequency [
21,
63].
The involvement of RHOB, TLR1, TNFRSF1A, LYN, CASP1, PRKCD, FCGR2A, and IFNGR1 in neuroinflammation promotes temporal lobe epileptogenicity (Fig.
3). Leukocyte expression of these neuroinflammation generating genes is uniquely activated in TLE patients with low seizure frequency when compared to patients with high seizure frequency in this study.
Oxidative stress (RAC2, NCF2, HMOX1)
Oxidative stress associated enzymes and markers are elevated in surgically resected human epileptogenic brain tissue and are involved in epileptogenesis [
64,
65]. Upregulation of leukocyte NCF2, RAC2 and HMOX1 expression in this study is predictive of low temporal lobe epilepsy seizure frequency, while downregulation of these genes predicts high seizure frequency. All of these genes play a role in oxidative stress and can be implicated in the process of redox imbalance (Fig.
4).
RAC2 (Family Small GTPase 2) is an important neutrophil component of the NADPH oxidase (NOX) multi-protein complex and regulates generation of oxidative stress (OS) reactive oxidative species (ROS), superoxide (O
2−) and hydrogen peroxide (H
2O
2), which are demonstrated in animal and in vitro models of epilepsy [
64,
66]. NOX activation generates high levels of neuronal and glial lipid peroxidation in human epileptogenic CA1 neuronal hippocampal plasma membranes [
66].
The NCF2 transcript protein, p67phox, is an important cytosolic component of the NADPH oxidase (NOX) multi-protein complex which works in concert with RAC2 to maximize electron transport [
66]. Phagocyte NADPH oxidase regulates major immune system pathways including type 1 interferon signaling, inflammasomes and autophagy as well as the generation of oxidative stress (OS), reactive oxidative species (ROS), superoxide (O
2−) and hydrogen peroxide (H
2O
2) [
66]. There is emerging evidence that temporal lobe epilepsy is a disease of redox imbalance involving reactive oxygen species pathways [
64,
67,
68]. As such, temporal lobe epilepsy ictal electrocorticographic and clinical seizure onset appear to be epiphenomena, preceded by more fundamental physiologic perturbations [
64,
67,
68]. For instance, in the cat hippocampal penicillin model of epilepsy, Ammon’s horn neuronal mitochondrial redox changes precede electrographic ictal onset by over 3 min [
67]. This pre-ictal decline in NADH is due to NADH oxidation and suggests that hippocampal neuronal energy change contributes to epileptogenesis [
67]. Similarly, HMOX1 upregulation is also associated with mitochondrial oxidative stress, but also with NF-κB-associated neuronal toxicity and glial-related neurodegeneration [
69]. HMOX1 is known to be a ubiquitous and redox-sensitive inducible stress protein [
65]. Additionally, mitochondrial astrocytic integrity is undermined by HMOX1 which mediates gliopathy, lowering the threshold of neighboring neuronal elements to oxidative injury [
70]. In cultured rat astroglia, miRNAs, regulated by HMOX1, cause mitochondria-dependent apoptosis and cell death, enhance TNFα biosynthesis which may worsen bioenergetic insufficiency, and compromise neural oxidative phosphorylation [
70]. HMOX1 dysregulation is also significant in its role of iron-induced production of free radicals [
71]. Iron generates free radicals which damage cell membranes via lipid peroxidation producing neuronal injury, increased extracellular glutamate and excitation as well as decreased GABA-A receptor function and inhibition, and epileptic discharges [
72]. In a rat model of cortical and neuronal hemin toxicity, neuronal cell death is prevented by treatment with the anticonvulsant, Valproic acid, which inhibits hemin toxicity by downregulation of HMOX1 [
73]. This is further evidenced by the pentylenetetrazole (PTZ) kindled mouse model of epilepsy which produces significant neuronal cell loss associated with oxidative stress, lipid peroxidation and enhanced hippocampal HMOX1 expression [
74].
Heme Oxygenase 1 (HMOX1) catabolizes heme to carbon monoxide, biliverdin and free ferrous iron (Fe2 +), the latter inducing production of free radicals, lipid peroxidation and oxidative stress producing neuronal injury and epileptic discharges [
71,
72]. Receptors of the Fc (fragment crystallizable) region of immunoglobulins (FcRs), including FcγRIIA, regulate antibody-dependent immune-complex-triggered inflammation through superoxide anions, lipoprotein oxidation and neurotoxicity and are associated with immune complex-mediated seizures [
59,
60]. The hippocampal microglia Src protein tyrosine kinase (SFK), LYN, upregulates nitro-oxidative stressors with production of electrographic seizures and increased epileptiform spiking [
75].
Enzymes involved in oxidative stress and oxidative stress markers have been shown to be elevated in surgically resected human epileptogenic brain tissue, supporting a redox imbalance hypothesis of human temporal lobe epileptogenesis [
64]. Also, oxidative stress activates inflammasomes which are important drivers of inflammation, neuronal degeneration, and neurodegenerative disease, including epilepsy [
76]. NCF2, RAC2 and HMOX1 activity all promote epileptogenicity through mitochondrial oxidative stress, neuronal lipid peroxidation, and neuronal and glial toxicity, all of which are consistent with the redox imbalance hypothesis of temporal lobe epilepsy (Fig.
4).
Glutamate is the main excitatory neurotransmitter in the brain and, through ionotropic glutamate receptors, controls most excitatory neurotransmission [
77]. N-methyl-D-aspartate (NMDA) and 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl) propanoic acid (AMPA) ionotropic glutamate receptors (a) mediate neuronal hyperexcitability and are present in human epilepsy and in kindling models of epilepsy and (b) initiate epileptiform discharges, mediate fast synaptic excitation, and are important in the spread and synchronization of epileptic activity, respectively [
36,
77‐
79]. Patients with temporal lobe epilepsy demonstrate reduced GABAergic- associated inhibition within the epileptic hippocampus and decreased numbers of GABA-ergic interneurons and GABA-ergic terminals and absent functional inhibition contributing to spontaneous recurrent seizures [
80,
81] (Fig.
5).
As a Src protein tyrosine kinase (SFK), another gene found in this study, LYN.
(LYN Proto-Oncogene, Src Family Tyrosine Kinase), upregulates NMDA receptor mediated neuronal hyperexcitability [
43,
45,
82]. These SFKs upregulate NMDA receptor (NMDAR) mediated neuronal hyperexcitability through phosphorylation and augmentation of NMDARs [
36,
82,
83]. LYN activation of P2X4 receptors produces microglial brain-derived neurotrophic factor (BDNF) secretion increasing NMDAR currents affecting neuronal plasticity and excitability [
42,
44]. Conversely SFK inhibition decreases in vitro hippocampal epileptiform discharge frequency and in vivo duration and number of seizures in mice [
36,
45]. Also in the NMDR signaling pathway is another gene found in our study, MYD88 (MYD88 Innate Immune Signal Transduction Adaptor). MYD88 is an intracellular adapter protein involved in the IL-1β signaling cascade which causes increased Tyr 1472-phosphorylation of the NR2B subunit of the NMDA receptor (NMDAR) and pharmacologic inhibition of MYD88 prevents the proconvulsant effects of IL-1β [
84,
85]. Inhibition of hippocampal MYD88 reduces N-methyl-D-aspartate receptor NR1 subunit expression and increases glutamate transporter 1 expression, protecting pyramidal neurons from apoptosis [
86]. Interestingly, MYD88 may also be responsible for NMDA receptor expression [
86].
Another gene seen in our study related to Glutamate/GABA-Mediated Excitotoxicity is CHP1. CHP1 (calcineurin homologous protein 1) upregulation is shown to inhibit NHE1 (Na
+/H
+ exchanger). Loss of the NHE1 (Na
+/H
+ exchanger 1) isoform in turn inhibits GABA-loaded vesicle release and causes central nervous system hyperexcitability ultimately resulting in epilepsy [
87,
88]. CHP subfamily targeting of NHEs is complex, depending on the applied stimulus and cellular environment; activity of the NHE1 isoform may be accentuated or inhibited by binding to CHP1 [
89]. NHE1, the principal target of CHP1, confers resistance to apoptosis and loss of NHE1, a neuronal plasma membrane constituent of the hippocampus and cortex, increases central nervous system excitability and produces epilepsy [
87,
88,
90]. Mice which lack NHE1 also demonstrate increased hippocampal neuronal excitability and higher Na
+ channel subtype I current density [
89,
91].
Lastly, the gene TNFRSF1A, in addition to being pro-inflammatory, also activates AMPA signaling creating pro-convulsant conditions. Specifically, TNFα alters the molecular stoichiometry and modulates the homeostatic synaptic scaling of post-synaptic AMPARs (AMPA (alpha-amino-3-hydroxy-5-methyl- 4-isoxazolepropionic acid receptors)-glutamate receptors), increases hippocampal neuronal cell surface GluA-AMPARs and increases excitatory synaptic activity of TNFRSF1A receptors [
92‐
94]. Post-synaptic AMPAR down-scaling and decreased overall AMPAR scaling capacity have been suggested as explanations for maintaining high and low seizure thresholds in temporal lobe epilepsy patients with low and high seizure frequency, respectively [
12].