Authors: Hayder M. Al-Kuraishy, Majid S. Jabir, Ali I. Al-Gareeb, Ali K. Albuhadily, Daniel J. Klionsky, Mayyadah F. Rafeeq
Categories: Review, Autophagy, autophagy inducers, epilepsy, neurological disease, seizure
Source: Autophagy
Authors: Hayder M. Al-Kuraishy, Majid S. Jabir, Ali I. Al-Gareeb, Ali K. Albuhadily, Daniel J. Klionsky, Mayyadah F. Rafeeq
Epilepsy is a neurological disease characterized by repeated unprovoked seizure. Epilepsy is controlled by anti-epileptic drugs (AEDs); however, one third of epileptic patients have symptoms that are not controlled by AEDs in a condition called refractory epilepsy. Dysregulation of macroautophagy/autophagy is involved in the pathogenesis of epilepsy. Autophagy prevents the development and progression of epilepsy through regulating the balance between inhibitory and excitatory neurotransmitters. Induction of autophagy and autophagy-related proteins could be a novel therapeutic strategy in the management of epilepsy. Despite the protective role of autophagy against epileptogenesis and epilepsy, its role in status epilepticus is perplexing and might reflect its nature as a double-edged sword. Autophagy inducers play a critical role in reducing seizure frequency and severity, and could be an adjuvant treatment in the management of epilepsy. However, autophagy inhibitors also have an anticonvulsant effect. Therefore, the aim of the present mini-review is to discuss the potential role of autophagy in the pathogenesis of epileptogenesis and epilepsy, and how autophagy modulators affect epileptogenesis and epilepsy.
Abbreviations: AD: Alzheimer disease; AEDs: antiepileptic drugs; AMPK: adenosine monophosphate-activated protein kinase; ER: endoplasmic reticulum; GABA: gamma aminobutyric acid; HCQ: hydroxycholoroquine; IP3: inositol 1,4,5-trisphosphate; NSAID: non-steroidal anti-inflammatory drug; PI3K: phosphoinositide 3-kinase; ROS: reactive oxygen species; SE: status epilepticus; PTZ: pentylenetetrazole; TLE: temporal lobe epilepsy; TSC: tuberous sclerosis complex
Epilepsy is a chronic neurological disease characterized by unprovoked repeated seizures, which correspond to an abnormal hyper-synchronous neuronal discharge from a definite brain region [1,2]. In ancient Greece, epilepsy was regarded as a form of spiritual, so called “sacred”, disease [3]. In addition, epileptic seizure was listed in the Hammurabi Code from 1790 BC [4]. Jean-Martin Charcot in the 19^th^ century discovered that epilepsy was misdiagnosed with mental disorders [5], and antiepileptic drugs (AEDs) including phenobarbital and phenytoin were introduced in the management of epilepsy in 1912 and 1938, respectively [6]. Despite the use of AEDs approximately 33% of epileptic patients have symptoms that are not controlled by these drugs [1,2]. Epilepsy affects approximately 1% of the general population internationally as of 2020 [7]. Most, ~80%, of epileptic cases universally occur in developing countries, and it is more common in the elderly population [8]. Approximately 5–10% of older people have a seizure by the age of 80 with a subsequent 40–50% increased chance for a second seizure [9].
The fundamental mechanism of epileptic seizure is due to epileptogenesis, which happens due to an inequity between excitatory and inhibitory neurotransmitters [10]. The pathogenesis of epileptogenesis is unknown, although mutation of voltage-gated Na^+^, Ca^2+^ and K^+^ channels is implicated in the development of neuronal hyper-excitability and decreased seizure threshold [11]. Mutation of the Na^+^ channel gene SCN8A (sodium voltage-gated channel alpha subunit 8) is accompanied by the development of epileptogenesis. These changes lead to reduction of inhibitory gamma aminobutyric acid (GABA) and/or an increase of excitatory glutamate neurotransmitters [10,12] (Figure 1). Figure 1.Pathophysiology of epilepsy. GABA and glutamate act as inhibitory and excitatory molecules, respectively. GABA induces neuronal hyperpolarization by increasing the conductance of the Cl^−^ channel, whereas glutamate excites the neurons by inducing the opening of the excitatory Na^+^ channel.
Notably, epilepsy is classified according to the underlying causes into primary (idiopathic) and secondary. The causes of primary epilepsy are not well identified, although genetic mutation in the neuronal ion channels are a key part of the proposed mechanism [13]. Nevertheless, secondary epilepsy is the result of diverse causes such as head trauma, brain infection, tumors and neurodegenerative disorders [14]. Epileptogenesis and epilepsy are linked with alteration of many cellular and subcellular processes. In particular, neuronal autophagy is implicated in the pathogenesis of epileptogenesis and the development of epilepsy [15,16]. It has been stated that the dysregulation of autophagy is intricately involved in the pathogenesis of epilepsy [17,18]. Neuronal autophagy regulates the expression of membrane GABA receptors and preserves neuronal excitatory/inhibitory balance. For example, the GABAA receptor trafficking function of GABARAP (GABA type A receptor-associated protein) family proteins is compromised due to autophagy deficiency [15,16]. In addition, glutamate stimulates autophagy via nicotinic acid adenine dinucleotide phosphate (NAADP), which acts as a second messenger for glutamate in neuronal and glial cells, and controls the cell metabolism in the central nervous system [15,16]. Therefore, autophagy is not merely a housekeeping process; instead, autophagy may be dynamically regulated in different neuronal compartments and contribute to specialized neuronal functions.
Consequently, the aim of the present mini-review is to discuss the potential role of autophagy in the pathogenesis of epileptogenesis and epilepsy, and how autophagy modulators (activators and inhibitors) affect epileptogenesis and epilepsy.
Autophagy is a specific cellular process to deliver different cytoplasmic misfolded proteins, lipids and damaged or superfluous organelles to the lysosomes for degradation and elimination [19]. There are several types of autophagy including microautophagy, macroautophagy and chaperone-mediated autophagy/CMA, and these can be characterized in part with regard to their mechanism in terms of membrane dynamics and whether they are selective or nonselective [20]. Macroautophagy is the best characterized and hence referred to as autophagy. In this process cytoplasmic components are engulfed within a transient double-membrane structure, the phagophore; this sequestering compartment matures into an autophagosome, which subsequently fuses with a lysosome (or first with an endosome) to deliver its contents for degradation and recycling [21]. Fusion of the external membrane of the autophagosome with the lysosome creates an autolysosome, which releases the breakdown products via permeases back into the cytosol [20,21]. The autolysosome then undergoes a process of autophagic lysosome reformation/ALR to regenerate the lysosome [22]. Therefore, autophagy participates in the renewal of cell constituents by recycling cytoplasmic macromolecules to form energy-rich compounds according to the bio-energetic demands [22]. Different autophagy-related proteins such as ATG5 (autophagy related 5), ATG12, ATG16L1 (autophagy related 16 like 1), and MAP1LC3/LC3 (microtubule associated protein 1 light chain 3) are involved in forming nascent autophagosomes from phagophores, a process that is triggered by the ULK1 (unc-51 like autophagy activating kinase 1) kinase and phosphatidylinositol 3-kinase (PtdIns3K) complexes [23] (Figure 2). Figure 2.The morphological intermediates of autophagy. The phagophore nucleates at the omegasome on the endoplasmic reticulum membrane (not depicted for simplicity) with additional membrane being supplied by the Golgi apparatus, endosomes and/or the plasma membrane. Expansion of the phagophore leads to formation of the double-membrane autophagosome, which fuses with a lysosome to form an autolysosome. PtdIns3K, phosphatidylinositol 3-kinase.
In general, autophagy has anti-aging and cytoprotective effects in many organ systems such as the central nervous system, liver, kidney, and heart [24,25]. With regard to neurodegeneration, the key role of autophagy is achieved through the induction of this degradative pathway to eliminate misfolded proteins and neurodegenerative-associated proteins such as MAPT/tau (microtubule associated protein tau) and SNCA (synuclein alpha) [26]. As well, autophagy promotes mitochondrial function and prevents the development of oxidative stress [27]. Autophagy is also involved in the degradation of infectious agents and attenuates various viral and bacterial infections [28]. In addition, autophagy has a critical role against proliferation, progression and metastasis of cancer cells [29]. Autophagy regulates cell death by controlling apoptosis and proapoptotic/apoptotic signaling [30]. Furthermore, autophagy regulates the immune response [31], type II diabetes [32], cardiomyopathy [33], and neurodegenerative diseases [34–36] (Figure 3). Figure 3.The protective role of autophagy. Autophagy attenuates (inhibitory bar) the development of neurodegenerative diseases, cancer, type II diabetes, and aging by regulating the immune response and cell death; however, in some diseases, autophagy has a double-edged role and can also promote (arrow) the disease.
Different autophagy signaling is involved in the regulation of neuronal autophagy (Table 1). The initiation of the autophagy process is mediated by two main complexes that include ULK1-ATG13-ATG101-RB1CC1 (RB1 inducible coiled-coil1) and the PtdIns3K lipid kinase complex that is comprised of PIK3C3/VPS34 (phosphatidylinositol 3-kinase catalytic subunit type 3), PIK3R4/VPS15 (phosphoinositide-3-kinase regulatory subunit 4), BECN1 (beclin 1), ATG14 and NRBF2 (nuclear receptor binding factor 2) [37].Table 1.Autophagy signaling.SignalingEffectsRef.Autophagy initiatorsMTORAMPKIGFTSCTP53IMPAIP3Induce formation of autophagosomesInhibits autophagyInduces autophagy by inhibition of MTOR, and through activation of ULK1 and SIRT1Inhibits autophagyTSC1-TSC2 inhibits MTORC1 and induces autophagyNuclear TP53 induces autophagy fluxIMPA induces autophagy independent of MTORIP3 receptors block autophagy through inhibition of BECN1[37][38][40,41][51][53][54][57][60]
Starvation or caloric restriction together with growth factors and tyrosine kinase receptors control the autophagic flux through regulation of diverse autophagy signals that play critical roles in the creation of autophagosomes. Starvation inhibits MTOR; however, when nutrients are replenished the MTOR pathway is reactivated with a significant reduction of autophagosomes [38]. Starvation-induced autophagy is mediated in part by attenuating the interaction between the autophagy inhibitory protein BCL2 (BCL2 apoptosis regulator) and BECN1, which involves additional negative factors such as BAX (BCL2 associated X, apoptosis regulator) and BAK1 (BCL2 antagonist/killer 1), whereas the BCL2-BECN1 interaction is abolished via phosphorylation of BCL2 by MAPK8/JNK1 (mitogen-activated protein kinase 8) promoting autophagy [46]. Caloric restriction activates the expression of adenosine monophosphate-activated protein kinase (AMPK), which induces direct activation of autophagy. AMPK induces autophagy by inhibition of MTOR, and through activation of ULK1 [39]. In addition, AMPK can activate the autophagy process through induction of SIRT1 (sirtuin 1) [40,47]. In the aging process, SIRT1 expression is reduced leading to a reduction in autophagy activity [48]. SIRT1-induced deacetylation activates FOXO3 (forkhead box O3) which is a specific transcription factor that reduces the formation of reactive oxygen species (ROS) and neuronal apoptosis [49]. Moreover, proteins such as BECN1, ATG5 and ATG7 are downregulated while inositol 1,4,5-trisphosphate (IP3) is upregulated during aging and neurodegenerative diseases.
MTOR is a conserved serine/threonine kinase that senses cellular energy and nutrition and is the central negative regulator of autophagy [50,51]. During over-nutrition, MTOR signaling is activated by growth factors and mitogens, which stimulate the formation of MTOR complex 1 (MTORC1) and MTORC2 resulting in the suppression of autophagy. Conversely, during starvation and caloric restriction the expression of growth factor proteins is decreased with subsequent reduction in the formation and activation of the MTOR complexes [51]. MTORC1 consists of RPTOR (regulatory associated protein of MTOR complex 1) a scaffold protein that is required for the correct subcellular localization of MTORC1, MLST8 (MTOR associated protein, LST8 homolog), which associates with the catalytic domain of MTOR and stabilizes the kinase activation loop, and the two inhibitory subunits AKT1S1/PRAS40 (AKT1 substrate 1) and DEPTOR (DEP domain containing MTOR interacting protein) [52]. Notably, MTORC2 shares some components with MTORC1 including the core kinase protein MTOR as well as MLST8 and DEPTOR. However, instead of RPTOR, MTORC2 contains RICTOR (RPTOR independent companion of MTOR complex 2), MAPKAP1/SIN1 (MAPK associated protein 1), PRR5/Protor1 (proline rich 5) and PRR5L (proline rich 5 like) [53]. MTORC1 and MTORC2 can also be distinguished based on their sensitivity to rapamycin which is much more inhibitory for MTORC1 [54].
Consistently, genetic deletion or pharmacological inhibition of MTOR is considered as the mainstay in the activation of autophagy in the management of different diseases such as cancer [51], with substantial focus on the MTOR inhibitor rapamycin or its derivatives [55]. Conversely, growth factors such as IGF (insulin like growth factor) via activation of IGF receptors enhance the activity of receptor tyrosine kinases and AKT (AKT serine/threonine kinase) leading to stimulation of the MTOR pathway and suppression of autophagy [41]. Particularly, AKT inhibits TSC1 (TSC complex subunit 1), which is an important component of the phosphoinositide 3-kinase (PI3K)-AKT-MTOR signaling pathway and plays a critical role in cell proliferation and development in conjunction with different regulatory molecules [56]. The TSC1-TSC2 complex inhibits MTORC1, which otherwise inhibits autophagy through phosphorylation of EIF4EBP1 (eukaryotic translation initiation factor 4E binding protein 1), eukaryotic translation initiation factor 4F (EIF4F) and RPS6KB1 (ribosomal protein S6 kinase B1) proteins [42].
Furthermore, TP53 (tumor protein p53) regulates the MTOR pathway and autophagy flux [43]. Of interest, genotoxic stress triggers TP53, which promotes activation of AMPK and PTEN (phosphatase and tensin homolog) and inhibits AKT signaling [57]. In contrast, suppression of TP53 also activates autophagy [58] because this protein can stimulate or inhibit autophagy dependent on its subcellular location. Furthermore, inhibition of IMPA (inositol monophosphatase) independent of MTOR induces autophagy [44]; inhibition of IMPA by carbamazepine, lithium, and valproate activates autophagy in various neuropsychiatric disorders [59]. Particularly, IP3 inhibits autophagy through activation of the endoplasmic reticulum (ER) ITPR/IP3 receptor (inositol 1,4,5-trisphosphate receptor), which induces the release of Ca^2+^ leading to the stimulation of the calpain pathway and inhibition of autophagy [60]. Accordingly, reduction of IP3 levels reduces ER Ca^2+^ release with the subsequent activation of autophagy through an AMPK-dependent pathway [60]. In addition, ITPRs block autophagy through inhibition of BECN1 [45]. These findings indicate that a range of proteins are engaged in the modulation of autophagy, which is highly dysregulated in aging [61] (Figure 4).
Figure 4.Regulation of autophagy. AKT, which is an important component of the PI3K-AKT-MTOR signaling pathway, inhibits TSC1-TSC2 and plays a critical role in cell proliferation and development in conjunction with different regulatory molecules. The TSC1-TSC2 complex inhibits MTORC1, which otherwise blocks autophagy through phosphorylation of EIF4EBP1, EIF4F and RPS6KB1. Caloric restriction activates AMPK, which induces direct activation of autophagy. AMPK also induces autophagy by inhibition of MTOR via SIRT1. Growth factors such as IGF activate their receptors, enhancing the activity of receptor tyrosine kinases and AKT leading to stimulation of the MTOR pathway and suppression of autophagy.
Epileptogenesis is a chronic neurological process by which a normal brain network is functionally changed toward increased epileptic seizure susceptibility and augmented possibility to generate unprovoked recurrent seizures [62]. Conventionally, epileptogenesis has been regarded as a latent period between epileptogenic insult and the development of the first clinical epileptic seizure [62]. Numerous studies highlight that the severity and frequency of unprovoked epileptic seizures are exaggerated following the first unprovoked episode [63] signifying that epileptogenesis is continuous even after the development of unprovoked epileptic seizure. The causes for the development of epileptogenesis are brain injury, ischemic stroke, and status epilepticus [62,63]. The mechanisms of epileptogenesis may be loss of a brain inhibitory circuit and/or synaptic reorganization with positive feedback activation of glutamatergic neurotransmission [64,65]. At the molecular level, many signaling pathways such as those involving MTOR, PI3K-AKT, IGF1, JAK (Janus kinase)-STAT3 (signal transducer and activator of transcription 3), and TGFB (transforming growth factor beta) are altered and involved in the development and progression of epileptogenesis. In addition, alterations of genes intricately involved in synaptic vesicle trafficking and glial oxidative stress are affected during the development of epileptogenesis [66]. Therefore, epileptogenesis is similar to epilepsy, and targeting of epileptogenesis may prevent the development and progression of epilepsy. However, AEDs do not prevent epileptogenesis.
Autophagy is implicated in the pathogenesis of epileptogenesis [67] (Table 2). Exaggerated MTOR pathway signaling (“MTORopathy”) leads to the inhibition of neuronal autophagy and acceleration of neuronal injury in epileptogenesis [67]. Findings from a preclinical study demonstrated that an aberrant overactivated MTOR signaling pathway due to suppression of TSC1 and PTEN is implicated in the development of epileptogenesis by inhibition of neuronal autophagy [68]. In addition, mutations of the TSC1 and TSC2 genes trigger the activation of the MTOR signaling pathway, which induces the progression of epileptogenesis through inhibition of autophagy [69]. Likewise, deletion of the Atg7 gene in mouse forebrain neurons promotes the progression of epileptogenesis [68]. Kulikov et al. [70] found that activation of neuronal autophagy during the initial phase of epileptogenesis has a neuroprotective effect in rats with experimental audiogenic seizure. Therefore, impaired neuronal autophagy is associated with the development and progression of epileptogenesis, and activation of autophagy could be effective against epileptogenesis. However, TGFB, which induces autophagy, is implicated in the pathogenesis of epileptogenesis [71,72]. Furthermore, PI3K-AKT, IGF1, and JAK-STAT3, which are all implicated in epileptogenesis [66] also affect autophagy [73]. Hence, the exact role of autophagy in epileptogenesis is mainly related to the specific neuronal signaling pathway that is altered during epileptogenesis. In addition, defective neuronal autophagy in epileptogenesis may be primary or secondary events that need to be elucidated by future studies.Table 2.The association between autophagy and epileptogenesis.Study typeFindingsRef.PreclinicalAn aberrant overactivated MTOR signaling pathway isimplicated in the development of epileptogenesis by inhibitionof neuronal autophagy in mice[68]PreclinicalDeletion of the Atg7 gene in mouse forebrain neurons promotesthe progression of epileptogenesis[68]PreclinicalTSC1 and TSC2 gene mutations trigger the activation of theMTOR signaling pathway, which induces the progression ofepileptogenesis through inhibition of autophagy[69]PreclinicalActivation of neuronal autophagy during the initial phase ofepileptogenesis has a neuroprotective effect in rats withexperimental seizure[70]PreclinicalTGFB, which induces autophagy, is implicated in the pathogenesis of epileptogenesis[71,72]PreclinicalPI3K-AKT, IGF1, and JAK-STAT3, which inhibit autophagy are implicated in epileptogenesis[66,73]
Autophagy plays a critical role in the pathogenesis of epilepsy through modulation of neurotransmitter synthesis and release. For example, autophagy promotes the balance between inhibitory GABA and excitatory glutamate by providing amino acids required for synthesis of GABA and glutamate [74]. Neuronal autophagy improves clustering of GABA receptors and thereby modulates the synaptic plasticity and membrane seizure threshold [75]. Defective autophagy is associated with a decrease in the activity of GABAergic neurotransmission [76]. For example, impairment of neuronal autophagy reduces the functional activity of GABAergic interneurons with the development of peripheral neuropathy [76]. Of note, distortion of brain GABAergic interneurons due to defective neuronal autophagy enhances the development of epileptic seizure [77]. Defective GABAergic interneurons not only are responsible for inhibition of neuronal excitability but also regulate neural network activity within the activated microcircuit during epileptic seizure [77]. Moreover, presynaptic autophagy controls the synthesis and release of many neurotransmitters [78]. Knockout of the Atg5 gene promotes the release of excitatory glutamate independent of synaptic density in primary cultured neurons [79]. Also, epilepsy is connected with overactivation of glutamate neurotransmission and excitotoxicity, which induces impairment of autophagy in hippocampal neurons [80], and autophagy activators can mitigate neuronal excitotoxicity in epilepsy [81]. Conversely, Okerlund et al. [82] demonstrated that autophagy induction prompts the release of excitatory neurotransmitters. Therefore, the effects of autophagy on the inhibitory and excitatory neurotransmitters seem modulatory.
The link between autophagy and epilepsy was confirmed by numerous studies. Autophagy dysfunction is allied with the development of epilepsy, as deficiency of ATG7 and WIPI1 are linked with spontaneous epileptic seizure [15,16]. In addition, defective autophagy induces the development of epileptic seizure in animal and human studies [83,84] and the initiation of autophagy and expression of autophagy-related proteins such as ATG7, LC3, and BECN1 by endothelial progenitor cells could be a novel therapeutic strategy in the management of epilepsy [18]. Using lymphoblastoid cell lines derived from subjects with static encephalopathy showed that a deficiency of WDR45/WIPI4 (WD repeat domain 45) is associated with neurodegenerative disorder in humans [15]. Defective autophagy promotes the development and progression of epileptic seizure by reducing the elimination of misfolded proteins. In particular, in a rare familial case of generalized epilepsy with febrile seizures plus, a mutant GABAA receptor subunit exhibits abnormally slow degradation and the formation of protein aggregates, similar to classic neurodegenerative disorders [15]. Although the mechanism of this protein aggregation is not established, this mutant GABAA receptor is at least partially degraded by lysosomes, suggesting the potential involvement of impaired autophagy.
Inhibition of mitochondrial autophagy by 3-methyladenine/3-MA impairs elimination of damaged mitochondria and increases the vulnerability for epilepsy [85]. Altered mitochondrial dynamics can trigger the development of epileptic seizure by affecting VPS13D (vacuolar protein sorting 13 homolog D) and autophagy. In a rat epilepsy model, knockdown of the Vps13d gene decreases seizure latency but increases seizure frequency [86]. Thus, VPS13D by affecting mitochondrial dynamics and autophagy can modulates epileptic seizure.
Mitochondria are both the source and target of metabolic and homeostatic dysfunction during seizures and epilepsy. Ca^2+^ excess is another key candidate for these changes, but the contribution of intracellular calcium stores and particularly the ER in Ca^2+^ excess during seizures and epilepsy remains understudied. Moreover, mitochondrial and ER-Ca^2+^ stores are intimately linked with each other [85,86]; however, the detailed nature of this interconnection in seizures and epilepsy remains an open question. It is known that Ca^2+^ together with ROS induces cell death during seizure activity. However, the precise sources of ROS involved remain a matter of debate. It is likely that different sources are active at different time points during seizures and epilepsy. The role of the NADPH oxidase with a focus on the different subtypes is of pressing importance, and such investigations are ideally performed in knockout animals, because current evidence on the role of NADPH oxidase in seizures and epilepsy relies on pharmacological manipulation [85,86]. This creates a problem because NADPH oxidase inhibitors are rarely isoform selective and often not even NADPH oxidase specific. Further characterization of this interaction will help to design ideal drug targets and allow for combinations of approaches to combat seizure-induced ROS. The intertwined role of Ca^2+^, ROS, and NADPH oxidase contribute to one of the main events leading to cell death and continuous seizures during seizure activity and thus contributes to epilepsy and epilepsy comorbidities.
In addition, epileptic seizure leads to inhibition of neuronal autophagy, which further worsens the symptoms [87]. Defective autophagy and exaggeration of MTOR signaling are linked with the development of tuberous sclerosis complex (TSC) and focal cortical dysplasia that are often associated with resistant epilepsy in children [84]. Autophagy activity is extremely inhibited in the forebrain neurons of patients with TSC [83]. In addition, deletion of the Atg7 gene in mouse forebrain neurons enhances seizure progression due to defective neuronal activity [83]. Importantly, mutations of TSC1-TSC2 lead to overactivation of the MTOR signaling pathway and the development of resistant epilepsy in animal models and humans. Thus, inhibition of an aberrant or overactivated MTOR signaling pathway could be effective in TSC patients [83]. Likewise, an exaggerated MTOR signaling pathway may progress in response to different immunological disorders. This change leads to synaptic dysfunction and progression of epileptic seizure [88]. It has been suggested that cell clearance systems including autophagy are considered as a bridge between neuro-immunity and the development of synaptic impairment by activating MTOR signaling with subsequent inhibition of neuronal autophagy in different autoimmune diseases [88]. Therefore, autophagy activators may reduce epilepsy risk in autoimmune disease [89].
The interaction between the apoptosis-related protein BCL2 and BECN1 is dysregulated during epileptic seizure [90]. An experimental study showed that BECN1 and LC3 levels are increased, whereas the BCL2 level is reduced within 48 h following seizure in rats [90]. This finding indicates that autophagy is activated following epilepsy. In addition, the BECN1-interacting protein AMBRA1 (autophagy and beclin 1 regulator 1) dissociates from BCL2 following autophagy induction leading to the activation of autophagy [90]. Therefore, autophagy activation following epilepsy could be a compensatory mechanism to limit neuronal loss. In fact, suppression of autophagy promotes seizure and neuronal apoptosis [91]. In addition, different preclinical studies illustrate that the LC3-II:LC3-I ratio (an indicator of autophagy induction) and BECN1 expression are augmented in an animal model of epilepsy [92,93]. Remarkably, induced autophagy in response to oxidative stress contributes to neuronal cell death after seizure [94]. Inhibition of oxidative stress by antioxidants considerably attenuates the autophagic response in pilocarpine-induced epilepsy [94]. These observations indicate that autophagy activity in epilepsy is altered in response to the effect of oxidative stress. Mounting evidence indicates that oxidative stress plays a critical role in different forms of neuronal death induced by epileptic seizures. The brain is particularly vulnerable to damage caused by oxidative stress, and an increase in oxidative stress biomarkers is found in various epilepsy types [94]. However, the fundamental molecular mechanism of neuronal death caused by oxidative stress in epileptic seizure is not fully elucidated.
Defective autophagy is common in early Lafora disease, which is a rare autosomal recessive disorder characterized by neurodegeneration, cognitive impairment and resistant myoclonic epilepsy. In add Lafora is characterized by the accumulation of insoluble abnormal glycogen deposits in the brain and peripheral tissues. Mutations in the EPM2A (EPM2A glucan phosphatase, laforin), and NHLRC1/EPM2B/malin (NHL repeat containing E3 ubiquitin protein ligase 1) genes contribute to the pathogenesis of Lafora disease. EPM2A and NHLRC1 form a complex in which EPM2A recruits the substrates to be ubiquitinated by NHLRC1 [95]. The EPM2A-NHLRC1 complex interacts with the PtdIns3K complex, increasing its activity, which induces autophagy activation [96].
Conversely, activated neuronal autophagy is exaggerated in status epilepticus as confirmed by many preclinical studies. Expression of brain LC3-II is augmented in mice with experimental epileptic seizure [97]. The LC3-II level is augmented comparable with ATG7 in the hippocampus after status epilepticus in mice [98]. Consistently, alteration of neuronal autophagy during epileptic seizure is mediated by MCOLN1/TRPML1 (mucolipin TRP cation channel 1), an ion channel that regulates the process of autophagy flux [99]. MCOLN1 is upregulated following experimental epileptic seizure leading to mitochondrial injury, oxidative stress, and neuronal apoptosis [99]. Consistently, activation of inhibitory neuronal autophagy by zinc oxide increases the severity of epileptic seizure by inducing an imbalance between inhibitory and excitatory neurotransmitters [100]. However, intensification of neuronal autophagy in acute epileptic seizure might be a compensatory mechanism to limit neuronal loss and support rearrangement of neurons and synaptic instability during seizure and status epilepticus [67]. Furthermore, the study of neuronal autophagy during epileptic seizure reveals that autophagy levels are reduced, and the incidence of epileptic seizure is significantly reduced after administration of rapamycin to upregulate autophagy [67]. Thus, the decreased level of neuronal autophagy during the latency period may be a possible mechanism for the increased susceptibility to epilepsy.
Furthermore, oxidative stress is implicated in the pathogenesis of epilepsy. Extreme production of ROS and the development of oxidative stress enhances neuronal hyperexcitability and initiates epileptic seizure [101]. The critical role of oxidative stress is not identical among different forms of epilepsy [102,103]. Furthermore, epileptic seizure and status epilepticus contribute to progressive neuronal injury via induction of oxidative injury of DNA, and membrane lipid and protein peroxidation by decreasing the production of ATP [104]. Many studies established that mitochondrial oxidative stress is augmented in chronic epilepsy and subsequent to status epilepticus [104,105]. Consistently, kainic-acid induced seizure stimulates hippocampal mitochondrial dysfunction and oxidative stress [102]. These observations suggest a common relationship between epilepsy and oxidative stress. Besides, oxidative stress affects the capacity of neuronal autophagy. Indeed, high oxidative stress triggers the activation of neuronal autophagy, although moderate levels of ROS activate only mitophagy [106,107]. Oxidative stress-induced autophagy is mediated by activation of many signaling pathways such as those involving MAPK/ERK, MAPK/JNK, and MAPK/p38 [108]. Similarly, high ROS levels via the activation of TSC2 promote the expression of mRNA encoding AMPK components, which induces stimulation of autophagy through inhibition of MTORC1 [109]. These findings highlight the fact that epilepsy-induced oxidative stress is responsible for activation of neuronal autophagy. In this manner, the activated neuronal autophagy could be a compensatory mechanism to limit oxidative stress-induced neuronal injury by eliminating damaged proteins and organelles to prevent further oxidative damage [110].
In summary, dysregulation of neuronal autophagy is intricately involved in the pathogenesis of epilepsy. Loss of function of PTEN and TSC1 due to gene mutations augments MTORC1 with subsequent inhibition of neuronal autophagy. Similarly, mutation of genes in Lafora disease reduces the expression of TSC2 with subsequent activation of MTOR. These alterations lead to the impairment of neuronal autophagy and the development of epilepsy. In turn, epilepsy leads to ATP exhaustion, ion channel imbalance, inflammation, and oxidative stress with progressive induction of autophagy. Interestingly, excessive uncontrolled autophagy activation is pro-epileptic; however, moderately activated neuronal autophagy has an antiepileptic effect. Therefore, neuronal autophagy has a double-edged effect in the development and progression of epilepsy (Figure 5). Figure 5.Autophagy and epilepsy. Loss of PTEN or TSC1 augments MTORC1 with inhibition of neuronal autophagy. In Lafora disease a reduction of the expression of TSC2 leads to activation of MTOR. These alterations induce impairment of neuronal autophagy and the development of epilepsy. Consecutively, epilepsy leads to ATP exhaustion, ion channel imbalance, inflammation, and oxidative stress with progressive induction of autophagy. Excessive uncontrolled autophagy activation is pro-epileptic, whereas moderately activated neuronal autophagy has an antiepileptic effect.
Many signaling pathways that affect the autophagy flux are involved in the pathogenesis of epilepsy (Table 3).Table 3.The association between autophagy signaling and epilepsy.Study typeFindingsRef.ClinicalMTOR overactivationis associated with inhibition ofautophagy and the development of refractory epilepsy[111]PreclinicalMutations of the TSC1 and TSC2 genes result in MTORhyperactivity and the development of tuberous sclerosiscomplex, which is often associated with refractory epilepsy[112]PreclinicalAMPK activates mitophagy, thereby eliminating damagedmitochondria, promoting cellular homeostasis and inhibitingepileptic seizure[113]PreclinicalGSK3B, which inhibits neuronal autophagy, triggers thedevelopment of epilepsy[114]PreclinicalThe expression of neuronal mitochondrial FOXO3, whichinduces autophagy is amplified in patients with TLE comparedto healthy controls[115]PreclinicalSIRT1 activates FOXO3, which triggers autophagy andsupports cell survival[116]PreclinicalPLD inhibits autophagy through activation of MTOR,inhibition of AMPK and through inhibition of the interactionbetween BECN1 and PIK3C3/VPS34[117,118]PreclinicalTP53 expression is correlated with epileptic seizure in animalmodels and TLE[119,120]
Normally, the MTOR pathway is critical in controlling neurogenesis, synaptic plasticity, neuronal development and excitability [121,122]. During brain development, the MTOR-mediated signaling pathway plays a critical role in the process of neuronal and glial differentiation and the conservation of the neural stem cells. The aberrations in the activity of MTOR and its downstream signaling result in defects of brain developmental processes causing many brain disorders, such as autism, pediatric brain tumors, epileptic seizure, mental retardation and learning disability [121,122]. However, excessive MTOR activity is linked with the development of temporal lobe epilepsy (TLE), genetic and acquired epilepsy, and Lafora disease [75,123]. Especially, MTORC1 is largely implicated in the pathogenesis of epileptogenesis and resistant epilepsy [123]. However, the fundamental mechanisms for induction of MTOR overactivation in epilepsy are not well elucidated. Of note, genetic deletion of the Tac1 (tachykinin 1) and Pten genes in forebrain neurons induces activation of the MTOR pathway, induction of epileptic seizure and inhibition of neuronal autophagy in mice [124]. Therefore, inhibition of the MTOR pathway diminishes seizure severity through induction of autophagy [121]. Mounting evidence from many studies highlights that the MTOR inhibitor rapamycin could be effective in the management of refractory epilepsy by attenuating the progression of epileptogenesis and the development of epilepsy [125–128].
Recently, MTOR-induced intractable epilepsy was found to be mediated by activation of EIF4E, which inhibits neuronal autophagy. Therefore, EIF4E inhibitors such as the antidiabetic metformin could be effective in treating refractory epilepsy [129]. In addition, mutations in genes involved in the MTOR signaling pathway result in the development of focal cortical dysplasia, which is associated with pediatric refractory epilepsy [111]. In focal cortical dysplasia, the severity of epileptic seizure is correlated with the density of brain dysmorphic neurons. Therefore, use of senolytic drugs such as dasatinib, which eliminate senescent cells, decreases the severity and seizure frequency in a transgenic mouse model [111]. Consequently, the autophagy and MTOR signaling pathways are interrelated in the development of epileptogenesis and epilepsy. Thus, pharmacological modulation of autophagy through an MTOR-dependent signaling pathway could provide a favorable outcome in the management of epilepsy [128]. Therefore, MTOR and its downstream effectors are implicated in the pathogenesis of epileptogenesis and epilepsy either by direct effect on the neurons or indirectly by inhibiting neuronal autophagy.
While there remains more to learn and understand about the role of the MTOR pathway in epilepsy, as well as the underlying cause of seizures in epilepsy, intervention along this pathway may lead to a reduction in seizure frequency, if not complete seizure freedom. This would be of great value for refractory epilepsy and may result in considerable improvements in quality of life and overall cognitive development.
TSC1 is an important component of the prosurvival PI3K-AKT-MTOR signaling pathway involved in cell growth, development, proliferation, survival, and induction of autophagy [130]. TSC1 has a tumor suppressive effect against different cancer types by regulating various signaling molecules. TSC2 plays an important role to control cell size in response to energy inadequacy and to protect cells from apoptosis [131]. The TSC1-TSC2 complex plays a crucial role in an evolutionarily conserved signaling pathway that regulates cell growth. Moreover, the TSC1-TSC2 complex coordinates and finely tunes a system in the central nervous system that has typical roles under diverse conditions, depending on cell type, stage of development, subcellular localization, and neural connectivity [132]. The TSC1-TSC2 protein complex negatively regulates the MTORC1 signaling pathway and induces activation of neuronal autophagy by activating AMPK [133]. Furthermore, dysregulation of the TSC1-TSC2 protein complex is implicated in the development of epilepsy. Mutations of the TSC1 and TSC2 genes provoke MTOR hyperactivity and the development of tuberous sclerosis complex, which is often associated with refractory epilepsy [112]. Notably, 75%-90% of patients with tuberous sclerosis complex have severe epilepsy at any age. Approximately 30% of children with tuberous sclerosis complex present with infantile spasm. Subsequently, multifocal seizures develop that are resistant to the effects of AEDs [112]. These findings support the hypothesis that autophagy induction by the TSC1-TSC2 protein complex is essential in preventing the development and progression of epilepsy.
That TSC may have MTORC1-independent functions in multiple areas of neural development and function is becoming clear. To accelerate progress in this area, it will be important to generate animal models that more closely replicate human disease. Major gaps in our knowledge include the the genetic and non-genetic modifiers of the TSC genes that account for the remarkable variability of expression within the human population; cell-type- and subcellular-location-specific roles of TSC1-TSC2 and their effectors; MTORC1-independent aspects of TSC regulation of neuronal function; the interplay between the different neurological symptoms of TSC disease; the relationship between neuronal energetics and the TSC-MTOR pathway. Studies that address such questions will shed light on the interaction between the TSC1 and TSC2 genes, the environment, and neurodevelopment in epilepsy.
AMPK is a conserved serine/threonine protein kinase that regulates metabolism by acting as an energy sensor. AMPK activates autophagy by direct inhibition of MTORC1 or indirectly through modulating the expression of ATG proteins [113]. In addition, AMPK activates mitophagy thereby eliminating damaged mitochondria and promoting cellular homeostasis [113]. Furthermore, AMPK advances synaptic plasticity and long-term potentiation by inhibiting neuronal hyper-excitability, regulation of ion channels and the development of epilepsy [134]. AMPK activation increases seizure threshold through modulation of MTORC1 and PPARGC1A/PGC-1α (PPARG coactivator 1 alpha) signaling [135]. Importantly, AMPK activators such as metformin are effective in treating refractory epilepsy by attenuating epileptogenesis [136]. Moreover, the AMPK signaling pathway is activated under a fasting state and during intermittent starvation. Therefore, caloric restriction and a ketogenic diet, by improving the brain AMPK signaling pathway, could be effective in the management of drug-resistant epilepsy [137,138]. The ketogenic diet is a high-fat, adequate protein and low carbohydrate diet that is indicated in the treatment of obesity, refractory epilepsy and neurodegenerative diseases such as Alzheimer disease [139]. The ketogenic diet provokes fasting-like states, which regulate the central and peripheral metabolism by inducing the activation of autophagy through an AMPK-dependent mechanism [139]. As well, a ketogenic diet regulates synaptic plasticity by inhibiting the development of oxidative stress and neuroinflammation [139,140]. A systematic review and meta-analysis observed that a ketogenic diet is associated with a reduction in seizure frequency by more than 50% and is effective in treating pediatric refractory epilepsy [140]. Likewise, caloric restriction attenuates the frequency and severity of epileptic seizures by inhibiting the development and progression of oxidative stress, neuroinflammation, and neurodegeneration through activation of the AMPK, and inhibition of the MTOR, signaling pathways [141].
Furthermore, AMPK activation through agents such as metformin has shown promising antiepileptic effects in various preclinical and clinical settings. These effects are primarily mediated through the inhibition of the MTOR signaling pathway, and activation of the AMPK-PI3K-JUN pathway [141]. Despite the potential of AMPK-targeted therapies, several aspects warrant further exploration, including the detailed mechanisms of AMPK’s role in different brain regions, the impact of AMPK under various conditional circumstances such as neural injury and zinc toxicity, the long-term safety and efficacy of chronic metformin use in epilepsy treatment, and the potential benefits of combination therapy involving AMPK activators. Moreover, the efficacy of AMPK activators in refractory epilepsy remains an open question. Therefore, direct activation of the AMPK signaling pathway by activators or indirectly by a ketogenic diet and caloric restriction can reduce the severity of intractable epilepsy by different mechanisms including autophagy activation.
GSK (glycogen synthase kinase) is a serine/threonine kinase that controls diverse cellular pathways such as cell proliferation, metabolism, differentiation, and apoptosis [142]. In addition, GSK promotes cellular anabolic and catabolic pathways by modulating glycogen synthase. There are two forms of GSK including GSK3A, which is distributed in certain brain regions, and GSK3B, which is ubiquitously distributed throughout the brain [142]. Aberrant activation of GSK3B is involved in the pathogenesis of epileptogenesis, epilepsy and neurodegenerative diseases [128]. Furthermore, epileptic seizure increases the expression of GSK3B which further exacerbates epileptic neuropathology [128]. It has been proposed that GSK3A could be a key factor that drives epileptogenesis in AD by interrelating with the APP (amyloid beta precursor protein) and MAPT/tau proteins. Besides, seizures may also participate to the progression of AD through GSK3A. In this way, GSK3A might be intricately involved in initiating a vicious cycle between AD and seizures [143]. Furthermore, mutant GSK3B increases synaptic excitability and contributes in the pathogenesis of epileptogenesis and epilepsy by dysregulating the sensitivity of neuronal ion channels [144]. Engel et al. [145] illustrated that overexpression of GSK3B augments hippocampal neuropathology by inducing neurodegeneration in mice with experimental status epilepticus. Surprisingly, decreasing GSK3B activity, either via overexpression of a dominant negative GSK3B or through the use of specific GSK3B inhibitors, also exacerbates hippocampal damage and increases seizure severity during status epilepticus [145], signifying that the brain has restricted tolerance for modulation of GSK3B activity in the setting of epileptic brain injury. These findings caution against targeting GSK3B as a treatment strategy for epilepsy. Moreover, overexpression of GSK3B plays a role in the inhibition and attenuation of neuronal autophagy [146,147]. GSK3B inhibits neuronal autophagy through modulation of many signaling pathways including TFEB (transcription factor EB), EIF4A3 (eukaryotic translation initiation factor 4A3), MTORC1, AKT and ULK1 [114]. Therefore, the GSK signaling pathway plays a critical role in the pathogenesis of epilepsy by inhibiting neuronal autophagy.
FOXO3 (forkhead box O3) is a transcription factor involved in cell cycle arrest, DNA repair, cell metabolism, differentiation, angiogenesis, aging and oxidative detoxification. It is ubiquitously expressed in human cells, and regulated by microRNAs [49]. FOXO3 attenuates oxidative stress through inducing the expression of mitochondrial antioxidant enzymes [148]. Of note, aberrant expression and exaggeration of the FOXO3 signaling pathway is associated with development of TLE [115]. Findings from a preclinical study demonstrated that neuronal expression of FOXO3 is augmented in hippocampal neurons of an animal epilepsy model [115]. Consistently, the expression of neuronal mitochondrial FOXO3 is amplified in patients with TLE compared to healthy controls [115]. FOXO3 is regarded as a potent activator of autophagy to eliminate oxidative molecules. Notably, FOXO3 prompts the activation of autophagy in neural stem cells [149]. In response to starvation, FOXO3 expression is upregulated leading to the induction of autophagy by promoting the formation of autophagosomes, and FOXO3 knockdown reduces autophagosomes in skeletal muscles during starvation [150].
FOXO3 has a neuroprotective role and reduces seizure severity in acute central nervous system toxicity by regulating oxidative stress [151]. Consequently, higher expression of FOXO3 in epilepsy might be a compensatory mechanism to mitigate oxidative stress and associated neuronal injury. However, reduction in the expression of FOXO3 mRNA is associated with the development of Lafora disease by impairment of neuronal autophagy [114]. Supporting this finding, FOXO3 mediates the neuroprotective effect of a ketogenic diet in the management of refractory epilepsy [152]. In addition, many AEDs produce their effect by activating the FOXO3 signaling pathway. For example, lamotrigine induces the expression of FOXO3, which promotes autophagy and reduces the severity of epileptic seizure [153]. As well, lamotrigine has an antitumor effect by increasing the expression of FOXO3 [153]. Moreover, acute valproate exposure enhances the expression of Foxo3 mRNA in the hippocampus of young mice [154]. These findings indicate that the neuroprotective FOXO3 signaling pathway, which induces autophagy activation, is highly reduced in epilepsy. Thus, activation of the FOXO3 signaling pathway might be a therapeutic strategy in the management of resistant epilepsy through activation of neuronal autophagy.
SIRT1 is a transcription factor that regulates cell survival and metabolism by activating autophagy [155]. Therefore, overexpression is associated with longevity and reduces aging-mediated disorders by interacting with FOXO3. Downregulation of SIRT1 is linked with the development of different neurodegenerative diseases [155]. SIRT1 levels are reduced during aging [47,48]. In addition, the SIRT1 signaling pathway is highly dysregulated in epilepsy. However, the exact role of SIRT1 in epilepsy is multifaceted, as it shows an upsurge subsequent to induced status epilepticus in mice; however, suppression of SIRT1 signaling does not affect the frequency, duration, and severity of status epilepticus in mice without exacerbation of epileptic seizure [156]. Consistently, brain SIRT1 expression is augmented in epileptic seizure within 1 h of epilepsy onset [157,158] and reduced after 24 h from onset of status epilepticus [159]. The protective effect of SIRT1 against epileptogenesis and epilepsy is associated with diverse mechanisms including activation of autophagy and enhancement of mitochondrial function. Moreover, SIRT1 provokes the expression of PPARGC1A, which reduces mitochondrial dysfunction and precludes development of oxidative stress [160]. SIRT1 also inactivates TP53, which is involved in neuronal cell death [161]. Likewise, SIRT1 activates FOXO3, which triggers autophagy and supports cell survival [116]. Of interest, the anticonvulsant oxaprozin reduces the severity of pentylentetetrazole-induced seizure in rat by upregulating SIRT1 expression [162]. In addition, valproate has a neuroprotective effect against the progression of spinocerebellar ataxia by augmenting the SIRT1 signaling pathway in vivo [163]. Consequently, the expression of SIRT1 following status epilepticus appears to be a compensatory mechanism by inducing neuronal autophagy.
PLA2 (phospholipase A2) catalyzes the hydrolysis of membrane phospholipid to generate arachidonic acid [164]. Likewise, PLD (phospholipase D) catalyzes the hydrolysis of membrane phosphatidylcholine to generate choline and phosphatidic acid, which regulate many cellular functions [165]. PLA2 is activated during epileptic seizure, and results in blood-brain barrier injury by upregulating MMP (matrix metallopeptidase) [166,167]. In addition, PLA2 upregulates ABCB1/P-glycoprotein (ATP binding cassette subfamily B member 1) in blood-brain barrier injury, increasing efflux of AEDs, and the development of drug-resistant epilepsy [164]. In particular, upregulation of cytosolic PLA2 in the hippocampus is associated with the development and severity of TLE in a rat model by increasing glutamatergic neurotransmission [166]. PLA2 triggers autophagy in patients with gouty arthritis [168]. Likewise, an in vitro study exposed that PLA2 promotes activation of macrophages [169]. PLD activity is increased within neurons and astrocytes in an epilepsy model [170]. PLD inhibits autophagy through activation of MTOR, inhibition of AMPK and through inhibition of the interaction between BECN1 and PIK3C3/VPS34 [117,118]. Consequently, activation of PLD and PLA2 during seizure may provide a noteworthy alteration of autophagy function.
IP3 inhibits autophagy through activation of ER ITPR/IP3 receptors [60]. IMPA inhibits autophagy [44]; therefore, inhibition of IP3 production or IMPA could improve neuronal autophagy. Lithium was previously and recently suggested to be effective in treating drug-resistant epilepsy by inhibiting the IP3-IMPA axis and promoting the induction of autophagy [171]. Equally, RAC1 (Rac family small GTPase 1), which is negatively regulated by MTOR, is involved in the pathogenesis of epileptogenesis [172]. Deletion of Rac1 activates the development of epilepsy by promoting neuronal hyper-excitability and decreasing of GABAergic inhibitory current in the hippocampus through inhibition of autophagy [172,173]. Indeed, rapamycin elicits the expression of RAC1. Therefore, defective RAC1 is associated with an exaggerated MTOR pathway and defective autophagy [87,174]. Furthermore, deletion of the Atg7 gene triggers recurrent seizure and the development of epilepsy in mice [83]. However, deletion of both Atg5 and Atg7 results in neurodegeneration without proof of epilepsy [175–177].
Furthermore, the TP53 gene, which encodes a transcription factor, controls the MTOR pathway and autophagy, and is intricately involved in the induction of neuronal apoptosis in response to oxidative stress [178]. In turn, oxidative stress promotes the expression of TP53 during epileptic seizure [43,179]. TP53 expression is correlated with epileptic seizure in animal models and TLE [119,120]. Thus, TP53 inhibitors might be effective in treating refractory epilepsy. Nonetheless, loss of TP53 worsens epilepsy following status epilepticus [180]. Consistently, kainic acid-induced seizure is more severe in TRP53-deficient mice compared to the wild type [180]. Consequently, the use of TP53-inhibitors for refractory epilepsy should be revised. Autophagy inhibits TP53 expression, and deficiency of ATG7 promotes TP53-dependent apoptosis [181]. Therefore, autophagy seems to play a critical role in the decrease of epilepsy by inhibiting the expression of TP53.
Overall, these findings highlight that dysregulation of autophagy signaling is implicated in the pathogenesis of epilepsy.
Of note, many AEDs through modulation of different signaling pathways affect the functional capacity of autophagy flux [59]. Valproic acid is an AED used in treating all types of epilepsy, bipolar disorders, and migraine headache. The mechanisms of action of valproic acid are blocking of voltage-gated sodium channel, increasing of synaptic GABA by inhibiting ABAT/GABA transaminase (4-aminobutyrate aminotransferase) and GABA uptake, and inhibition of neurotransmitter-induced hyperexcitability through suppression of phosphatidylinositol (3,4,5)-trisphosphate/PIP3 [182]. Valproic acid inhibits HDAC6 (histone deacetylase 6) and increases lysosomal acidification by modulating V-ATPase assembly in AD [183]. Valproic acid enhances clearance of amyloid peptide by activating neuronal autophagy [183]. Thereby, valproic acid could be effective in treating neurodegenerative diseases. Wang et al. [184] illustrated that induction of autophagy by valproic acid is associated with attenuation of pentylenetetrazole (PTZ)-induced epileptic seizure in rats. In addition, valproic acid activates autophagy through modulation of AKT-MTOR signaling in glioma [185].
Carbamazepine is a potent AED indicated in treating focal epilepsy, myoclonic epilepsy, peripheral neuropathy, and neuropsychiatric disorders. Carbamazepine acts by blocking voltage-gated sodium and voltage-gated calcium channels and inhibiting serotonin reuptake and neurotransmitter release [186]. Carbamazepine improves motor impairment in a mouse model of Machado-Joseph disease by increasing neuronal autophagy via AMPK-dependent activation [187]. Qianq et al. [188] illustrated that carbamazepine, by inducing neuronal autophagy, prevents hippocampal neuronal apoptosis in rats with experimental intractable epilepsy. In addition, carbamazepine mitigates Aβ neuropathology by activating neuronal autophagy in a mouse AD model [189]. Therefore, carbamazepine has a neuroprotective effect against epilepsy and neurodegenerative diseases by triggering neuronal autophagy.
Levetiracetam is an AED indicated in treating tonic-clonic, myoclonic and partial onset epileptic seizures. The exact mechanism by which levetiracetam acts to treat epilepsy is unknown; it does not inhibit voltage-dependent Na^+^ channels, GABAergic transmission, and glutamatergic receptors [190]. Levetiracetam mitigates cognitive impairment in an AD mouse model by activating neuronal autophagy through inhibiting GSK3B [191]. Shi et al. [192] found that the AEDs levetiracetam and topiramate mitigate behavioral deficits in APP transgenic mice by stimulating neuronal autophagy.
Furthermore, perampanel which is the first AED in the class of selective noncompetitive AMPA receptor antagonists has a neuroprotective effect by stimulating neuronal autophagy through regulating the expression of glutamate receptors [193]. Moreover, vagus nerve stimulation improves cerebral blood flow and restores autophagic flux [194]. Vagus nerve stimulation is effective in controlling epileptic seizure in animal models [195]. Analysis of clinical trials and prospective studies revealed a clinical benefit for the use of vagus nerve stimulation and deep brain stimulation in the management of refractory epilepsy.
Taken together, the efficacy of AEDs in the management of epilepsy may be mediated by activation of neuronal autophagy (Table 4).Table 4.Effects of AEDs on autophagy.AEDsEffectsRef.Valproic acidActivates neuronal autophagy[183]CarbamazepineImproves motor impairment in a mouse model of Machado-Joseph disease by promoting neuronal autophagy via AMPK-dependent activation[187]LevetiracetamMitigates cognitive impairment in an AD mouse model byactivating neuronal autophagy by inhibiting GSK3B[191]TopiramateMitigates behavioral deficit in APP transgenic mice bystimulating neuronal autophagy[192]PerampanelActivates neuronal autophagy through regulating theexpression of glutamate receptors[193]
Many autophagy inducers are effective in the alleviation of epilepsy (Table 5).Table 5.Role of autophagy inducers in epilepsy.Autophagy inducersEffectsRef.RapamycinHas an epileptostatic effect by interfering with theepileptogenesis process through inhibition of MTORC1 andactivating autophagy[196]LithiumStimulates autophagy by inhibiting the negative regulator IP3.However, lithium has pro-convulsive, anti-convulsive andneuroprotective properties[171,197]MetforminImproves cognitive function and reduces seizure risk bymodulating the AMPK-MTOR pathway[198,199]IbuprofenEffective in rats with PTZ-induced seizure by inducingautophagy[200,201]FenofibrateHas an anticonvulsive effect by activating neuronal PPARAand autophagy[202]
Rapamycin is a macrocyclic lactone antibiotic produced by Streptomyces hygroscopicus as an antifungal agent. Furthermore, rapamycin has multiple actions including anti-proliferative and immunosuppressive effects through modulation of MTOR and FKBP5/FK506-binding protein (FKBP prolyl isomerase 5) [203]. Administration of rapamycin in late-life mice extends lifespan; thus, rapamycin may have an antiaging effect [204]. The underlying mechanism for the neuroprotective effect of rapamycin against neurodegenerative diseases and epilepsy is mediated by autophagy activation [204]. Supporting this claim, rapamycin attenuates seizure severity and frequency in chronic kainic acid-induced epileptic seizure in mice [205]. Rapamycin has an epileptostatic effect as it interferes with the epileptogenesis process [196]. Therefore, direct administration of rapamycin into the hippocampus does not prevent further epileptic seizure following status epilepticus in animal models [205]. However, findings from a preclinical study highlight that chronic administration of rapamycin in rats subsequent to status epilepticus precludes the development of epileptic seizure by maintaining the integrity of the blood-brain barrier [196,206], although whether there is recurrence of epileptic seizure subsequent to the cessation of rapamycin treatment was not reported. In addition, rapamycin attenuates pilocarpine-induced epilepsy in mice by inhibiting the MTORC1 signaling pathway [207]. These results point out that rapamycin has anti-epileptogenesis but not antiepileptic effects [196].
Moreover, rapamycin is effective against TSC-induced seizure in animal and human studies [208,209]. Rapamycin reduces seizure frequency in patients with TSC by 25% [209]. As well, prolonged use of rapamycin for 6 months reduces seizure frequency by 56.25% in TSC patients with refractory epilepsy [210]. Mounting evidence from clinical studies specifies that rapamycin is effective in patients with TSC and can be used with AEDs [211]. Likewise, everolimus, a derivative of rapamycin, has been approved as an adjuvant treatment in the management of epilepsy [212]. Similarly, everolimus reduces seizure frequency by 73% in TSC patients [213]. Prolonged use of everolimus decreases seizure frequency and severity in different types of drug-resistant epilepsy [214]. The mechanism of action of rapamycin is related to the inhibition of MTORC1 [215] and activation of FKBP5 [203], which inhibits the MTOR pathway. FKBP5 regulates GABAergic neurons and the expression of glutamate receptors in astrocytes, thus decreasing the severity and frequency of epileptic seizure activity [216]. Furthermore, FKBP5 triggers autophagy and augments the effect of antidepressant agents [217]; these drugs induce autophagy by amplifying the expression of FKBP5 [217]. In addition, rapamycin has immunomodulatory effects and thereby reduces inflammatory reactions induced by epileptic seizure and status epilepticus [218]. Rapamycin, by inhibiting MTORC1, attenuates hyperactivation of excitatory circuits and the frequency of excitatory post-synaptic current and epileptiform activity in mice with experimental TLE [219]. Interestingly, rapamycin and other MTOR inhibitors prevent the development of neuroinflammation induced by epileptic seizure and status epilepticus in diverse autoimmune disorders such as encephalitis-induced seizure [220,221]. Thus, rapamycin and everolimus by inhibiting MTOR, activating the expression of FKBP5 and inducing neuronal autophagy can decrease seizure activity and the development of epilepsy.
Lithium is a natural element used as a mood-stabilizing agent in treating bipolar disorders. However, the underlying mechanism of lithium in the management of bipolar disorders remains elusive. Lithium improves synaptic plasticity by regulating the expression of microRNA and inhibiting the activity of GSK3B [222]. Of note, lithium stimulates autophagy by inhibiting the negative regulator IP3 [197]. Also, lithium has direct and indirect inhibitory effects on the expression and activity of GSK3B a negative regulator of autophagy [223]. Although preclinical studies detected that high-dose lithium inhibits autophagy, a low dose activates autophagy [224]. Furthermore, lithium has pro-convulsive, anti-convulsive and neuroprotective properties [171]. A longitudinal study revealed that the use of lithium in patients with bipolar disorders and comorbid epilepsy should be combined with AEDs to reduce the risk of epileptic seizure [225]. In pilocarpine-induced seizure in mice, administration of lithium at a low dose (10 mg/kg) reduces seizure severity, although a high dose of lithium (40 mg/kg) increases seizure severity [226]. Diverse clinical studies revealed that long-term use of lithium is linked with myoclonic, tonic-clonic and non-convulsive status epilepticus [227,228]. In contrast, an observational study involving 1028 patients treated with lithium showed no clinical evidence for seizure control [211]. Thus, lithium displays dose-dependent anti-seizure activity by modulating autophagy. Despite these findings, the exact role of lithium in epilepsy is complex and requires further studies.
Metformin is an insulin-sensitizing drug used as a first-line treatment in the management of diabetes [229]. Metformin controls blood glucose by inhibiting hepatic glycogenolysis, decreasing gluconeogenesis, and increasing peripheral glucose uptake through an AMPK-dependent mechanism [230–233]. In addition, metformin has pleiotropic effects such as anti-inflammatory, antioxidant, antithrombotic and atheroprotective effects via AMPK-dependent and -independent mechanisms [234–237]. In addition, metformin has neuroprotective effects against neurodegenerative disorders by regulating synaptic plasticity, oxidative stress and neuroinflammation [229]. Furthermore, preclinical studies confirmed that metformin improves cognitive function and reduces seizure risk by modulating the AMPK-MTOR pathway [198,199]. Metformin had been confirmed to inhibit epileptogenesis and epileptic seizure in PTZ-induced epileptic seizure by increasing the expression of neuronal AMPK which is downregulated during acute seizure and chronic epilepsy [238].
Particularly, treatment with metformin reduces the severity and duration of epileptic activity and tempers post-ictal depression [238]. It has been established that metformin decreases PTZ-induced epilepsy in mice [239]. As well, metformin attenuates status epilepticus and the progression of TLE in rats [240]. In addition, pretreatment with metformin inhibits inflammatory cytokines and increases the neuroprotective GRN/progranulin (granulin precursor) and anti-inflammatory cytokines in rats with experimental TLE [241]. A systematic review highlighted that metformin may be effective against the development and progression of epileptic seizure in animal model studies [198]. In a clinical setting, a randomized clinical trial showed that metformin lessens the frequency of epileptic seizure in children with TSC and Lafora disease [242,243].
These findings highlight that metformin could be effectual in the management of epilepsy. The potential ant-epileptic mechanism of metformin is not fully elucidated. However, metformin regulates the AMPK-MTOR signaling pathway, which affects neurotransmitter release and synaptic plasticity [244–246]. AMPK, which is activated by metformin, enhances catabolic and inhibits anabolic pathways [245]. In contrast, MTOR is triggered by high energy and inhibits catabolic pathways [246]. Stimulation of AMPK by metformin results in the inhibition of MTOR. In addition, metformin can constrain MTOR activation through an AMPK-independent pathway [247]. AMPK has a neuroprotective effect by increasing the expression of SLC2A1/GLUT1 (solute carrier family 2 member 1), which is expressed in astrocytes and regulates central glucose homeostasis [248,249]. Deletion of astrocyte SLC2A1/GLUT1 induces epileptic seizure in patients with SLC2A1/GLUT1 deficiency syndrome [249]. AMPK enhances glucose uptake and glycolysis of astrocytes by increasing translocation of membrane SLC2A1/GLUT1 [250]; however, overactivation of AMPK during brain ischemia has deleterious effects [251]. In brain ischemia, AMPK is activated in the astrocytes due to an upsurge of nitric oxide, which inhibits mitochondrial respiration and promotes glycolysis [251].
Furthermore, AMPK prompts the expression of PPARGC1A, which improves mitochondrial biogenesis and upregulates SIRT1 and FOXO3 [252,253]. AMPK also inhibits synthesis of fatty acids by inducing the expression of malonyl-CoA which inhibits fatty acid oxidation [252]. Metformin also improves glycolysis in astrocytes [254,255]. Furthermore, AMPK modulates the thalamic spike wave seizure in hypoglycemia-induced absence seizure. Of interest, administration of metformin potentiates seizure network activity by activating postsynaptic GABBR (gamma-aminobutyric acid type B receptor) in the thalamocortical neurons of rats [256]. Therefore, metformin, like other AEDs such as tiagabine and vigabatrin, can trigger absence seizure by promoting the availability of GABA [257]. Furthermore, metformin is effective against the development and progression of TLE but worsens the activity of absence seizure [258]. Predominantly, absence seizure is common in children where metformin is rarely used. Therefore, the antiepileptic effect of metformin appears to be indistinguishable to the effect of AEDs, which are effective for generalized but not for absence seizure.
Conversely, certain types of epilepsy are related to the upregulation of the MTOR signaling pathway, and suppression of this pathway by metformin attenuates the frequency and severity of epileptic seizure [259]. Consistently, rapamycin attenuates lipopolysaccharide-induced absence seizure in rats [260]; however, metformin, which inhibits the MTOR pathway, exacerbates absence seizure [256]. Consequently, MTOR inhibitors employ diverse mechanistic effects against seizure neuro-activity. In addition, not all MTOR inhibitors are effective against the development of epileptogenesis and progression of epilepsy [261]. For example, experimental MTOR inhibitors such as AZD8055 and PF4708671 are ineffective in mice with epilepsy [261]. In addition, vigabatrin, which inhibits the MTOR pathway, interrupts but does not prevent the occurrence of epileptic seizure in mice [261].
Concerning the effect of metformin on neuronal autophagy, many studies found that metformin enhances autophagy by modulating the AMPK-MTOR axis [262,263]. Metformin attenuates aging-induced inflammation by inhibiting inflammation and activating neuronal autophagy [263,264]. Also, metformin inhibits the development of status epilepticus by inducing neuronal autophagy [265].
These outcomes indicate that metformin has anti-seizure activity by activating AMPK signaling and constraining the MTOR signaling pathway with subsequent activation of neuronal autophagy.
Ibuprofen is a non-steroidal anti-inflammatory drug (NSAID) that is commonly used as an anti-inflammatory, anti-pyretic, and analgesic. Most studies have focused on the anti-inflammatory and anti-pyretic properties of ibuprofen during febrile convulsions. Ibuprofen is a nonselective inhibitor of PTGS/cyclooxygenase/COX (prostaglandin-endoperoxide synthase) [266]. In addition, ibuprofen has a neuroprotective effect by constraining PTGS and the pro-inflammatory cytokine IL18 (interleukin 18) [200]. Findings from a preclinical study demonstrated that ibuprofen is effective in rats with PTZ-induced seizure [200,201]. The possible anti-seizure effect of ibuprofen is linked to its anti-inflammatory effect and through induction of neuronal autophagy. Of interest, ibuprofen sensitizes CD44-expressing cells to the effect of HSP90 through induction of autophagy [267], although most NSAIDs are effective in decreasing epileptic seizure in experimental models [268]; however, the selective PTGS2/COX2 inhibitor celecoxib is more effective compared to the selective PTGS inhibitors in pilocarpine-induced epilepsy [269]. A cohort study involving epileptic patients revealed that co-administration of ibuprofen reduces the valproic plasma level by 7.5–30.6% within 1 week [267]. Therefore, the use of ibuprofen in epileptic patients taking valproic acid, should be considered with caution. Thus, ibuprofen by activating neuronal autophagy could be an effective adjuvant with AEDs in the management of epilepsy.
Collectively, autophagy inducers might be effective in the management of epilepsy, particularly the refractory type. Further preclinical trials and prospective studies are recommended to verify the exact role in epileptogenesis and the development of epilepsy.
Fenofibrate is a lipid-lowering drug used in the management of hypertriglyceridemia and mixed dyslipidemia. It acts by activating PPARA/PPAR-α (peroxisome proliferator activated receptor alpha) in the peripheral tissues [270–272]. In addition, fenofibrate has pleiotropic anti-inflammatory, antioxidant and atheroprotective effects via PPARA-dependent and -independent mechanisms [273]. Interestingly, fenofibrate has neuroprotective effects against neurodegenerative diseases by activating PPARA widely distributed throughout the central nervous system [274]. Fenofibrate exerts an anticonvulsive effect comparable to that of a ketogenic diet in PTZ-induced seizure in rats and other epileptic seizure models by activating neuronal PPARA [202]. In additioon, fenofibrate reduces the frequency and severity of nocturnal frontal lobe epilepsy by modulating the activity of CHRNA/nAChRα/nicotinic acetylcholine receptor α (cholinergic receptor nicotinic alpha) in mice via activation of PPARA [275]. Sarahian et al. [276] confirmed that fenofibrate by its antioxidant effect reduces the severity of PTZ-induced epileptic seizure in mice. Likewise, fenofibrate attenuates PTZ-induced epileptic seizure in mice by regulating the expression of the neuroprotective BDNF (brain derived neurotrophic factor) [277].
Moreover, fenofibrate activates autophagy by increasing the expression of SIRT1 and FGF21 (fibroblast growth factor 21) in diabetic mice [278]. Ouk et al. [279] showed that fenofibrate improves acute and chronic brain ischemia in animal models by activating neuronal autophagy. Interestingly, fenofibrate improves the autophagy-lysosomal pathway, which is impaired in AD and thereby increases clearance of misfolded proteins and attenuates AD neuropathology [280]. Fenofibrate activates neuronal autophagy by upregulating TFEB, a key activator for autophagy [280]. Therefore, fenofibrate could be effective in treating epilepsy by activating neuronal autophagy.
Overall, autophagy inducers seem to have neuroprotective effects against the severity and frequency of epileptic seizure in epilepsy.
Autophagy could possibly be repressed at any stage of autophagy flux. During the study of autophagy mechanisms, many agent inhibitors have been recognized that are used in animal models. However, most chemical inhibitors of autophagy are not completely specific, and researchers and clinicians should be cautious in deducing the findings obtained with the use of these compounds, especially regarding their dose and duration. Nonetheless, the role of autophagy inhibitors in epilepsy is documented in many studies (Table 6).Table 6.Role of autophagy inhibitors in epilepsy.Autophagy inhibitorsEffectsRef.NilotinibHas neuroprotective and anticonvulsant effects by inhibitingautophagy, attenuation of inflammation and oxidative stress[281]Ascorbic acidLong-term use of ascorbic acid reduces the severity of epilepticseizure in a rat model by decreasing neuroinflammation,oxidative stress and inhibiting autophagy[282]HydroxycholoroquineLow-dose HCQ has anti-seizure effects, whereas a high doseof HCQ has contributory effects[283]
Nilotinib is a second-generation tyrosine kinase inhibitor used in the management of chronic myeloid leukemia [284]. Indeed, non-receptor tyrosine kinases are implicated in the pathogenesis of epilepsy [285]. Non-receptor tyrosine kinases are interrelated with autophagy by activating MAPK and the JAK-STAT-PI3K and MTORC1 signaling pathways leading to the modulation of autophagy in response to cellular stress [286]. Therefore, nilotinib by inhibiting the pathological role of non-receptor tyrosine kinases can attenuate the pathogenesis of epilepsy [281]. Findings from an experimental study demonstrated that nilotinib had neuroprotective and anticonvulsant effects in PTZ-induce seizure in rats by inhibiting autophagy, and attenuation of inflammation and oxidative stress [281]. Autophagy can paradoxically progress to cell death with prolonged stress [287]. Oxidative stress induced by epileptic seizure may lead to profound activation of autophagy, which provokes neuronal apoptosis [281]. Therefore, inhibition of neuronal autophagy by nilotinib can protect the neurons from the effect of exaggerated autophagy. However, nilotinib reduces Parkinson disease neuropathology by eliminating SNCA/α-synuclein via activation of neuronal autophagy [288]. Therefore, nilotinib seems to function as an autophagy modulator rather than inhibitor, and its neuroprotective effect may be mediated by inhibition of the exaggerated activity of tyrosine kinase.
Ascorbic acid is a water-soluble vitamin that has antioxidant effects similar to tocopherol. It crosses the blood-brain barrier and accumulates in the brain [286]. Ascorbic acid attenuates neuronal injury in the hippocampus during epileptic seizure by reducing the progression of oxidative stress [286]. Long-term use of ascorbic acid reduces the severity of epileptic seizure in a rat model by decreasing neuroinflammation and oxidative stress [282]. Interestingly, a low brain concentration of ascorbic acid triggers the development of epileptic seizure in AD patients [289]. Similarly, brain ascorbic acid concentration is low in AD due to a defect in the neuronal ascorbic acid transporter in an APP transgenic mouse model [289]. Therefore, low plasma ascorbic acid predisposes for the development and progression of epilepsy. Hence, ascorbic acid could be effective as an adjuvant treatment with AEDs for severe epileptic seizure.
Furthermore, ascorbic acid affects the functional capacity of neuronal autophagy during epileptic seizure by inhibiting neuronal oxidative stress, which induces autophagy [290]. Therefore, ascorbic acid is regarded as an autophagy inhibitor and thereby can mitigate the severity of epileptic seizure-induced neuronal injury [290]. Scalise et al. [291] revealed that ascorbic acid attenuates the development of TLE in children with febrile seizure by reducing oxidative stress-induced autophagy activation. Therefore, the beneficial anti-seizure effect of ascorbic acid is mainly mediated by inhibition of neuronal autophagy.
Hydroxycholoroquine (HCQ) is an antimalarial drug that has potent anti-inflammatory effects against different immunoinflammatory diseases such as rheumatoid arthritis. It acts by increasing lysosomal pH and inhibits the autophagy-lysosomal pathway. In addition, HCQ inhibits the innate immune response by blocking TLR9 (toll like receptor 9) [292]. Autophagy inhibitors including HCQ can block the progression of cancer [293]. Furthermore, HCQ is implicated in the development of epileptic seizure. Low-dose HCQ has anti-seizure effects although a high dose of HCQ has a contributory effect [283] suggesting a dose-dependent effect of HCQ on neuronal membrane seizure threshold. Findings from an experimental study highlight that low-dose HCQ increases neuronal membrane seizure threshold, whereas the reverse occurs with a high dose of HCQ [294]. However, a systematic review illustrates no association between HCQ and risk of epileptic seizure [295]. Despite these findings, the exact association between HCQ and epileptic seizure still requires further research.
Taken together, autophagy inducers seem to be more appropriate than autophagy inhibitors in the management of epilepsy.
Epilepsy is a neurological disease characterized by repeated seizure. Epilepsy is controlled by AEDs in approximately 69% of the affected population, whereas the symptoms of the remaining epileptic patients are not controlled by AEDs and are referred to as refractory epilepsy. Dysregulation of autophagy is an integral pathway in the pathogenesis of epilepsy. Autophagy prevents the development and progression of epilepsy through regulating the balance between inhibitory GABA and excitatory glutamate. Induction of autophagy might be an innovative therapeutic strategy in the management of epilepsy. Despite the protective role of autophagy against epilepsy, its role in status epilepticus is perplexing and might be a double-edged sword, being either detrimental or harmful. Autophagy inducers such as rapamycin, metformin, and ibuprofen play a critical role in reducing seizure frequency and severity, and could be adjuvant treatments in the management of epilepsy and drug-resistant epilepsy. Conversely, autophagy inhibitors also have anticonvulsant effects. Therefore, the function of autophagy needs further studies to identify the precise role of this pathway in epileptogenesis and epilepsy.
Structural and functional integrity in neurons is more sensitive to changes in autophagy than that of non-neuronal cells due to the highly specialized compartments necessary for intercellular communications. Autophagy alterations have begun to be applied to the field of epileptogenesis besides neurodegenerative disorders. Recent studies have suggested that hyperactivation of the MTOR signaling pathway and abnormal autophagy activity occur in different epilepsy animal models and epilepsy-related patients, whereas inhibition of MTOR activity can ameliorate seizures in various epilepsy models. Genetic sequencing of multiple types of epilepsy patients and seizure models indicate that gene mutations in autophagy-related pathways may contribute to the occurrence of epilepsy. In addition, impaired autophagy is sufficient to induce epilepsy in rodent models, such as genetic inactivation of ATG7 in mice. However, it represents an underexplored research avenue for epilepsy. There are still many important concepts, principles, and issues to be addressed in this field. Promising directions involve working out the details, understanding how synapses signal membrane biogenesis and autophagy initiation, to uncover how neuronal autophagy and synaptic efficacy are cross-regulated in seizures; to uncover the interconnection of autophagy activity with antiepileptic drugs; and to define the diversity and dynamic nature of the autophagic cargo of different neuronal compartments in terms of their physiological states and epilepsy pathologies.
Therefore, dysregulation of neuronal autophagy is intricately involved in the pathogenesis of epilepsy. Inhibition of neuronal autophagy with subsequent activation of MTOR triggers the development of epilepsy. In turn, epilepsy leads to the induction of autophagy. Excessive uncontrolled autophagy activation is pro-epileptic; however, moderately activated neuronal autophagy has an antiepileptic effect. Consequently, neuronal autophagy has a double-edged-sword effect in the development and progression of epilepsy. Thus, additional studies are recommended in this regard and to fill in the gaps that exemplify this perplexing relationship.