Metformin Plus Caloric Restriction Show Anti-epileptic Effects Mediated by mTOR Pathway Inhibition

Caloric restriction (CR) has anti-epileptic effects in different animal models, at least partially due to inhibition of the mechanistic or mammalian target of rapamycin (mTOR) signaling pathway. Adenosine monophosphate-activated protein kinase (AMPK) inhibits mTOR cascade function if energy levels are low. Since hyper-activation of mTOR participates in epilepsy, its inhibition results in beneficial anti-convulsive effects. A way to attain this is to activate AMPK with metformin. The effects of metformin, alone or combined with CR, on the electrical kindling epilepsy model and the mTOR cascade in the hippocampus and the neocortex were studied. Combined metformin plus CR beneficially affected many kindling aspects, especially those relating to generalized convulsive seizures. Therefore, metformin plus CR could decrease measures of epileptic activity in patients with generalized convulsive seizures. Patients that are obese, overweight or that have metabolic syndrome in addition to having an epileptic disease are an ideal population for clinical trials to test the effectiveness of metformin plus CR.     

Brain-derived neurotrophic factor facilitates TrkB down-regulation and neuronal injury after status epilepticus in the rat hippocampus

Brain-derived neurotrophic factor (BDNF) is involved in many aspects of neuronal biology and hippocampal physiology. Status epilepticus (SE) is a condition in which prolonged seizures lead to neuronal degeneration. SE-induced in rodents serves as a model of Temporal Lobe Epilepsy with hippocampal sclerosis, the most frequent epilepsy in humans. We have recently described a strong correlation between TrkB decrease and p75ntr increase with neuronal degeneration ( Neuroscience 154:978, 2008). In this report, we report that local, acute intra-hippocampal infusion of function-blocking antibodies against BDNF prevented both early TrkB down-regulation and neuronal degeneration after SE. Conversely, the infusion of recombinant human BDNF protein after SE greatly increased neuronal degeneration. The inhibition of BDNF mRNA translation by the infusion of antisense oligonucleotides induced a rapid decrease of BDNF protein levels, and a delayed increase. If seizures were induced at the time endogenous BDNF was decreased, SE-induced neuronal damage was prevented. On the other hand, if seizures were induced at the time endogenous BDNF was increased, SE-induced neuronal damage was exacerbated. These results indicate that under a pathological condition BDNF exacerbates neuronal injury.     

Evaluation of Cannabidiol in Animal Seizure Models by the Epilepsy Therapy Screening Program (ETSP)

Cannabidiol (CBD) is a cannabinoid component of marijuana that has no significant activity at cannabinoid receptors or psychoactive effects. There is considerable interest in CBD as a therapy for epilepsy. Almost a third of epilepsy patients are not adequately controlled by clinically available anti-seizure drugs (ASDs). Initial studies appear to demonstrate that CBD preparations may be a useful treatment for pharmacoresistant epilepsy. The National Institute of Neurological Disorders and Stroke (NINDS) funded Epilepsy Therapy Screening Program (ETSP) investigated CBD in a battery of seizure models using a refocused screening protocol aimed at identifying pharmacotherapies to address the unmet need in pharmacoresistant epilepsy. Applying this new screening workflow, CBD was investigated in mouse 6 Hz 44 mA, maximal electroshock (MES), corneal kindling models and rat MES and lamotrigine-resistant amygdala kindling models. Following intraperitoneal (i.p.) pretreatment, CBD produced dose-dependent protection in the acute seizure models; mouse 6 Hz 44 mA (ED₅₀ 164 mg/kg), mouse MES (ED₅₀ 83.5 mg/kg) and rat MES (ED₅₀ 88.9 mg/kg). In chronic models, CBD produced dose-dependent protection in the corneal kindled mouse (ED₅₀ 119 mg/kg) but CBD (up to 300 mg/kg) was not protective in the lamotrigine-resistant amygdala kindled rat. Motor impairment assessed in conjunction with the acute seizure models showed that CBD exerted seizure protection at non-impairing doses. The ETSP investigation demonstrates that CBD exhibits anti-seizure properties in acute seizure models and the corneal kindled mouse. However, further preclinical and clinical studies are needed to determine the potential for CBD to address the unmet needs in pharmacoresistant epilepsy.     

Seizures Are Regulated by Ubiquitin-specific Peptidase 9 X-linked (USP9X), a De-Ubiquitinase

Epilepsy is a common disabling disease with complex, multifactorial genetic and environmental etiology. The small fraction of epilepsies subject to Mendelian inheritance offers key insight into epilepsy disease mechanisms; and pathologies brought on by mutations in a single gene can point the way to generalizable therapeutic strategies. Mutations in the PRICKLE genes can cause seizures in humans, zebrafish, mice, and flies, suggesting the seizure-suppression pathway is evolutionarily conserved. This pathway has never been targeted for novel anti-seizure treatments. Here, the mammalian PRICKLE-interactome was defined, identifying prickle-interacting proteins that localize to synapses and a novel interacting partner, USP9X, a substrate-specific de-ubiquitinase. PRICKLE and USP9X interact through their carboxy-termini; and USP9X de-ubiquitinates PRICKLE, protecting it from proteasomal degradation. In forebrain neurons of mice, USP9X deficiency reduced levels of Prickle2 protein. Genetic analysis suggests the same pathway regulates Prickle-mediated seizures. The seizure phenotype was suppressed in prickle mutant flies by the small-molecule USP9X inhibitor, Degrasyn/WP1130, or by reducing the dose of fat facets a USP9X orthologue. USP9X mutations were identified by resequencing a cohort of patients with epileptic encephalopathy, one patient harbored a de novo missense mutation and another a novel coding mutation. Both USP9X variants were outside the PRICKLE-interacting domain. These findings demonstrate that USP9X inhibition can suppress prickle-mediated seizure activity, and that USP9X variants may predispose to seizures. These studies point to a new target for anti-seizure therapy and illustrate the translational power of studying diseases in species across the evolutionary spectrum.     

Inhibition of Death-associated Protein Kinase 1 protects against Epileptic Seizures in mice

Epilepsy is a chronic encephalopathy and one of the most common neurological disorders. Death-associated protein kinase 1 (DAPK1) expression has been shown to be upregulated in the brains of human epilepsy patients compared with those of normal subjects. However, little is known about the impact of DAPK1 on epileptic seizure conditions. In this study, we aim to clarify whether and how DAPK1 is regulated in epilepsy and whether targeting DAPK1 expression or activity has a protective effect against epilepsy using seizure animal models. Here, we found that cortical and hippocampal DAPK1 activity but not DAPK1 expression was increased immediately after convulsive pentylenetetrazol (PTZ) exposure in mice. However, DAPK1 overexpression was found after chronic low-dose PTZ insults during the kindling paradigm. The suppression of DAPK1 expression by genetic knockout significantly reduced PTZ-induced seizure phenotypes and the development of kindled seizures. Moreover, pharmacological inhibition of DAPK1 activity exerted rapid antiepileptic effects in both acute and chronic epilepsy mouse models. Mechanistically, PTZ stimulated the phosphorylation of NR2B through DAPK1 activation. Combined together, these results suggest that DAPK1 regulation is a novel mechanism for the control of both acute and chronic epilepsy and provide new therapeutic strategies for the treatment of human epilepsy.     

The Search for New Screening Models of Pharmacoresistant Epilepsy: Is Induction of Acute Seizures in Epileptic Rodents a Suitable Approach?

Epilepsy, a prevalent neurological disease characterized by spontaneous recurrent seizures (SRS), is often refractory to treatment with anti-seizure drugs (ASDs), so that more effective ASDs are urgently needed. For this purpose, it would be important to develop, validate, and implement new animal models of pharmacoresistant epilepsy into drug discovery. Several chronic animal models with difficult-to-treat SRS do exist; however, most of these models are not suited for drug screening, because drug testing on SRS necessitates laborious video-EEG seizure monitoring. More recently, it was proposed that, instead of monitoring SRS, chemical or electrical induction of acute seizures in epileptic rodents may be used as a surrogate for testing the efficacy of novel ASDs against refractory SRS. Indeed, several ASDs were shown to lose their efficacy on acute seizures, when such seizures were induced by pentylenetetrazole (PTZ) in epileptic rather than nonepileptic rats, whereas this was not observed when using the maximal electroshock seizure test. Subsequent studies confirmed the loss of anti-seizure efficacy of valproate against PTZ-induced seizures in epileptic mice, but several other ASDs were more potent against PTZ in epileptic than nonepileptic mice. This was also observed when using the 6-Hz model of partial seizures in epileptic mice, in which the potency of levetiracetam, in particular, was markedly increased compared to nonepileptic animals. Overall, these observations suggest that performing acute seizure tests in epileptic rodents provides valuable information on the pharmacological profile of ASDs, in particular those with mechanisms inherent to disease-induced brain alterations. However, it appears that further work is needed to define optimal approaches for acute seizure induction and generation of epileptic/drug refractory animals that would permit reliable screening of new ASDs with improved potential to provide seizure control in patients with pharmacoresistant epilepsy.     

Brain-derived neurotrophic factor (BDNF)-induced synthesis of early growth response factor 3 (Egr3) controls the levels of type A GABA receptor alpha 4 subunits in hippocampal neurons

Altered function of gamma-aminobutyric acid type A receptors (GABA(A)Rs) in dentate granule cells of the hippocampus has been associated with temporal lobe epilepsy (TLE) in humans and in animal models of TLE. Such altered receptor function (including increased inhibition by zinc and lack of modulation by benzodiazepines) is related, in part, to changes in the mRNA levels of certain GABA(A)R subunits, including alpha4, and may play a role in epileptogenesis. The majority of GABA(A)Rs that contain alpha4 subunits are extra-synaptic due to lack of the gamma2 subunit and presence of delta. However, it has been hypothesized that seizure activity may result in expression of synaptic receptors with altered properties driven by an increased pool of alpha4 subunits. Results of our previous work suggests that signaling via protein kinase C (PKC) and early growth response factor 3 (Egr3) is the plasticity trigger for aberrant alpha4 subunit gene (GABRA4) expression after status epilepticus. We now report that brain derived neurotrophic factor (BDNF) is the endogenous signal that induces Egr3 expression via a PKC/MAPK-dependent pathway. Taken together with the fact that blockade of tyrosine kinase (Trk) neurotrophin receptors reduces basal GABRA4 promoter activity by 50%, our findings support a role for BDNF as the mediator of Egr3-induced GABRA4 regulation in developing neurons and epilepsy and, moreover, suggest that BDNF may alter inhibitory processing in the brain by regulating the balance between phasic and tonic inhibition.     

Hyperphosphorylated Tau is Implicated in Acquired Epilepsy and Neuropsychiatric Comorbidities

Epilepsy is a common group of neurological diseases. Acquired epilepsy can be caused by brain insults, such as trauma, infection or tumour, and followed by a latent period from several months to years before the emergence of recurrent spontaneous seizures. More than 50 % of epilepsy cases will develop chronic neurodegenerative, neurocognitive and neuropsychiatric comorbidities. It is important to understand the mechanisms by which a brain insult results in acquired epilepsy and comorbidities in order to identify targets for novel therapeutic interventions that may mitigate these outcomes. Recent studies have implicated the hyperphosphorylated tubulin-associated protein (tau) in rodent models of epilepsy and Alzheimer's disease, and in experimental and clinical studies of traumatic brain injury. This potentially represents a novel target to mitigate epilepsy and associated neurocognitive and psychiatric disorders post-brain injury. This article reviews the potential role of tau-based mechanisms in the pathophysiology of acquired epilepsy and its neurocognitive and neuropsychiatric comorbidities, and the potential to target these for novel disease-modifying treatments.     

Synchrotron X-ray microbeams: A promising tool for drug-resistant epilepsy treatment

Epilepsy is one of the most important neurological diseases. It concerns about 1% of the population worldwide. Despite the discovery of new molecules, one third of epileptic patients are resistant to anti-epileptic drugs and among them only a few can benefit from resective surgery. In this context, radiotherapy is an interesting alternative to the other treatments and several clinical devices exist (e.g., Gamma Knife^®). The European Synchrotron Radiation Facility offers the possibility to develop new methods of radiosurgery and to study their antiepileptic effects. Here, we discuss several studies that we performed recently to test and try to understand the antiepileptic effects of X-ray synchrotron microbeams in different animal models of epilepsy. We showed a decrease of seizures after Interlaced Microbeam Radiotherapy (IntMRT) of the somatosensory cortex, known as the seizure generator, in a genetic model of absence epilepsy. These antiepileptic effects were stable over 4 months and with low tissular and functional side-effects. The irradiated pyramidal neurons still displayed their physiological activity but did not synchronize anymore. We also obtained a lasting suppression of seizures after IntMRT of the dorsal hippocampus in a mouse model of mesiotemporal lobe epilepsy. However, an important variability of antiepileptic efficiency was observed probably due to the small size of the targeted structure. Despite these encouraging proofs-of-concepts, there is now a need to adapt IntMRT to other models of epilepsy in rodents which are close to refractory forms of epilepsy in human patients and to implement this approach to non-human primates, before moving to clinical trials.     

MicroRNA与癫痫的研究进展

癫痫是大脑神经元高度同步化异常放电所导致的短暂的大脑功能障碍的一种慢性疾病。癫痫的发病原因十分复杂,目前主要治疗方式是药物治疗,但仍然有30%左右的难治性癫痫患者依靠药物治疗未能控制癫痫发作,因此从分子角度研究癫痫的发病机制及治疗是近年来癫痫研究的热点。微小RNA(miRNA)在癫痫患者及癫痫动物模型海马组织中存在差异性表达,通过抑制miRNA的差异表达在一定程度上可以缓解癫痫的症状,这为癫痫的治疗开辟了新的途径和方向。因此随着miRNA与癫痫相关性研究深度的不断加深,有望能够为癫痫的诊断及治疗提供一个全新的思路。     

Mutations in prickle orthologs cause seizures in flies, mice, and humans

Epilepsy is heritable, yet few causative gene mutations have been identified, and thus far no human epilepsy gene mutations have been found to produce seizures in invertebrates. Here we show that mutations in prickle genes are associated with seizures in humans, mice, and flies. We identified human epilepsy patients with heterozygous mutations in either PRICKLE1 or PRICKLE2. In overexpression assays in zebrafish, prickle mutations resulted in aberrant prickle function. A seizure phenotype was present in the Prickle1-null mutant mouse, two Prickle1 point mutant (missense and nonsense) mice, and a Prickle2-null mutant mouse. Drosophila with prickle mutations displayed seizures that were responsive to anti-epileptic medication, and homozygous mutant embryos showed neuronal defects. These results suggest that prickle mutations have caused seizures throughout evolution.     

Disease-Modifying Effects of Neural Regeneration Peptide 2945 in the GAERS Model of Absence Epilepsy

Neurochemical Research - Epilepsy is a common neurological condition characterised by spontaneous recurrent seizures. Current anti-epileptic drugs are only effective and tolerated in ~70% of...     

Towards an integrated view of HCN channel role in epilepsy

Epilepsy is the third most common brain disorder and affects millions of people. Epilepsy is characterized by the occurrence of spontaneous seizures, that is, bursts of synchronous firing of large populations of neurons. These are believed to result from abnormal regulation of neuronal excitability that favors hypersynchrony. Among the intrinsic conductances that govern neuronal excitability, the hyperpolarization-activated current (I(h)) plays complex and important roles in the fine-tuning of both cellular and network activity. Not surprisingly, dysregulation of I(h) and/or of its conducting ion-channels (HCN) has been strongly implicated in various experimental models of epilepsy, as well as in human epilepsy. Here we provide an overview of recent findings on the distinct physiological roles played by I(h) in specific contexts, and the cellular mechanisms that underlie these functions, including the subunit make-up of the channels. We further discuss current knowledge of dysregulation of I(h) and HCN channels in epilepsy in light of the multifaceted functions of I(h) in the brain.     

Depolarization promotes survival of ciliary ganglion neurons by BDNF-dependent and -independent mechanisms

Membrane activity upregulates brain derived neurotrophic factor (BDNF) expression to coordinately support neuronal survival in many systems. In parasympathetic ciliary ganglion (CG) neurons, activity mimicked by KCl depolarization provides nearly full trophic support. While BDNF has been considered unable to influence CG neuronal survival, we now document its expression during CG development and show that low concentrations do support survival via high-affinity TrkB receptors. Furthermore, a contribution of BDNF to activity-induced trophic support was demonstrated by showing that KCl depolarization increased BDNF mRNA and protein in, and release of BDNF from, CG neuron cultures. Application of anti-BDNF blocking antibody or mitogen activated protein kinase (MAPK) kinase inhibitor, attenuated depolarization-supported survival, implicating canonical BDNF/TrkB signaling. Ca2+-Calmodulin kinase II (CaMKII) was also required since its inhibition combined with anti-BDNF or MAPK kinase inhibitor abolished or greatly reduced the trophic effects of depolarization. Membrane activity may thus support CG neuronal survival both by stimulating release of BDNF that binds high-affinity TrkB receptors to activate MAPK and by recruiting CaMKII. This mechanism could have relevance late in development in vivo as ganglionic transmission and the effectiveness of BDNF over other growth factors both increase.     

褪黑素的抑痫机制

癫痫(epilepsy)是临床上一种常见病、多发病,其发病机制尚不完全清楚。褪黑素(melatonin,MT)是一种神经内分泌激素,近年的研究表明,MT具有抑制癫痫发作的功能。MT抑制癫痫的作用机制研究也在不断开展。本文将从不同层面对MT抑痫机制的研究进展做一综述,为进一步的探索提供研究基础。     

HIV-1 Tat-mediated protein transduction of human brain creatine kinase into PC12 cells

Epilepsy is characterized by the presence of spontaneous episodes of abnormal neuronal discharges and its pathogenic mechanisms remain poorly understood. Recently, we found that the expression of creatine kinase (CK) was markedly decreased in an epilepsy animal model using proteomic analysis. A human CK gene was fused with a HIV-1 Tat peptide to generate an in-frame Tat-CK fusion protein. The purified Tat-CK fusion protein was efficiently transduced into PC12 cells in a time- and dose-dependent manner when added exogenously to culture media. Once inside the cells, the transduced Tat-CK fusion protein was stable for 48 h. Moreover, the Tat-CK fusion protein markedly increased endogenous CK activity levels within the cells. These results suggest that Tat-CK provides a strategy for the therapeutic delivery of proteins in various human diseases including the delivery of CK for potential epilepsy treatment.     

Validation of a Preclinical Drug Screening Platform for Pharmacoresistant Epilepsy

The successful identification of promising investigational therapies for the treatment of epilepsy can be credited to the use of numerous animal models of seizure and epilepsy for over 80 years. In this time, the maximal electroshock test in mice and rats, the subcutaneous pentylenetetrazol test in mice and rats, and more recently the 6 Hz assay in mice, have been utilized as primary models of electrically or chemically-evoked seizures in neurologically intact rodents. In addition, rodent kindling models, in which chronic network hyperexcitability has developed, have been used to identify new agents. It is clear that this traditional screening approach has greatly expanded the number of marketed drugs available to manage the symptomatic seizures associated with epilepsy. In spite of the numerous antiseizure drugs (ASDs) on the market today, the fact remains that nearly 30% of patients are resistant to these currently available medications. To address this unmet medical need, the National Institute of Neurological Disorders and Stroke (NINDS) Epilepsy Therapy Screening Program (ETSP) revised its approach to the early evaluation of investigational agents for the treatment of epilepsy in 2015 to include a focus on preclinical approaches to model pharmacoresistant seizures. This present report highlights the in vivo and in vitro findings associated with the initial pharmacological validation of this testing approach using a number of mechanistically diverse, commercially available antiseizure drugs, as well as several probe compounds that are of potential mechanistic interest to the clinical management of epilepsy.