Epilepsy is a chronic neurological disorder characterized by recurrent seizures, in which oxidative stress and neuroinflammation play central roles in driving disease progression and pharmacoresistance. Approximately 30-40 % of patients are resistant to current antiseizure medications, which suppress symptoms but do not prevent epilepsy development or modify its progression. There is an urgent need for therapies with true disease-modifying potential. TXM-CB3 (CB3), a thioredoxin-mimetic tripeptide, has been reported to modulate redox and inflammatory pathways. In this study, we evaluated the therapeutic potential of CB3 in preclinical models of temporal lobe epilepsy, focusing on its capacity to suppress seizures, preserve neuronal integrity, and mitigate epilepsy-associated behavioral impairments. We first examined CB3 in an in vitro model of low-Mg2+-induced epileptiform activity, where pretreatment with CB3 (50, 100 μM) attenuated oxidative activity and reduced proinflammatory cytokine expression (IL-6, IL-1β, TNF-α), while enhancing IL-10 levels. In vivo, early CB3 intervention (20 mg/kg/day, i.p.) following kainic acid-induced status epilepticus significantly delayed seizure onset, reduced seizure frequency and cumulative burden, and preserved hippocampal neuronal integrity. Treated animals also showed improved locomotor activity, reduced anxiety-like behavior, and better performance in spatial working memory tasks. In established chronic epilepsy, CB3 treatment (20 mg/kg/day, i.p.) produced a sustained reduction in recurrent seizure activity and seizure burden, with additional effects on anxiety-like behavior, though memory and learning deficits remained unchanged. Together, these findings highlight CB3's potential as a disease-modifying therapy. By reducing seizure recurrence, preserving neuronal integrity, and alleviating selected behavioral impairments, CB3 offers therapeutic benefits that extend beyond conventional ASMs and warrants further investigation for translation into clinical epilepsy treatment.

Fast-ripples (250-500 Hz) have been proposed as a promising biomarker in epilepsy, but their specificity remains unclear. In particular, it is uncertain whether they reflect chance coincident neural activity or distinctly generated pathological entities. We combined in silico simulations, neuronal cultures, the intra-hippocampal kainate rat model of hippocampal epilepsy, and human microwire recordings to investigate whether fast-ripples occur more frequently than expected by the chance aggregation of action potentials. Our simulations showed that chance aggregation can generate fast-ripples and that their incidence changes depending on brain state, an observation that we confirmed in our rodent data. The likelihood of exceeding chance depended on biological system complexity and vigilance state: fast-ripples in neuronal cultures did not surpass chance levels, whereas those in awake – but not sleeping – rodents did. Similarly, the incidence of fast-ripples in awake human recordings was ∼30% greater than expected by chance. As such, our findings suggest that most fast-ripples reflect stochastic network activity rather than distinctly generated pathological entities. This work does not rule out FRs as potential indicators of epileptogenic tissue, but it does challenge prevailing assumptions about their generation and specificity. Their higher prevalence in epileptogenic tissue is likely primarily due to increased excitation and/or neural synchronization, rather than peculiar abnormalities in network behavior.

Bi-allelic WWOX loss-of-function mutations underlie a spectrum of developmental and epileptic encephalopathies, including the severe WOREE syndrome and the milder SCAR12, for which no effective therapies exist. Gene replacement represents a compelling strategy; however, critical parameters for clinically translatable delivery remain undefined. Here, we establish a rationally optimized WWOX gene therapy framework using a severe Wwox-null mouse model. Systematic evaluation of promoter selection, cellular targeting, and vector configuration, dose, and timing identified neuron-restricted expression driven by the human synapsin I promoter as the most effective approach, yielding robust and durable phenotypic rescue compared to non-specific or oligodendrocyte-directed strategies. Removal of the WPRE element enabled controlled transgene expression and facilitated dose calibration within a clinically relevant range. An optimal AAV9-hSynI-WWOX dose restored survival, growth, metabolic function, behavior, and fertility to near wild-type levels. Therapeutic efficacy was associated with sustained reconstitution of WWOX across central and peripheral neural tissues, absence of off-target hepatic expression, enhanced myelination, and reduced neuroinflammation. Early neuronal hyperexcitability was suppressed following treatment. Importantly, efficacy was shown in an early postnatal therapeutic window (P1-P5), supporting durable rescue. Collectively, these findings define key design and dosing principles for translation of WWOX gene therapy to patients with developmental and epileptic encephalopathies.

OBJECTIVE:Epilepsy is a chronic neurological disorder characterized by recurrent seizures and frequent cognitive and psychiatric comorbidities. Although current antiseizure medications provide symptomatic relief, they fail to prevent or modify epileptogenesis. Heat shock protein 90 (Hsp90) is increasingly recognized as a regulator of neuroinflammatory and oxidative stress pathways implicated in seizure generation and disease progression. Here, we investigated the therapeutic potential of cemdomespib, a novel and selective Hsp90 inhibitor, across complementary preclinical models of epilepsy. METHODS:In vitro, cemdomespib was evaluated in the low-magnesium model of epileptiform activity for its effects on neuronal calcium dynamics, mitochondrial membrane stability, and reactive oxygen species (ROS) generation. In vivo, acute seizure protection was assessed in the pentylenetetrazol (PTZ) model, and antiepileptogenic efficacy was tested in the kainic acid-induced status epilepticus (KA-SE) model using chronic video-electrocorticographic recordings. Behavioral outcomes relevant to epilepsy-associated comorbidities, including anxiety-like behavior and exploratory activity, were also examined. RESULTS:Cemdomespib reduced epileptiform calcium oscillations, stabilized mitochondrial membrane potential, and suppressed ROS generation in vitro. In the PTZ model, 45% of pretreated animals were protected from seizures, and those that seized exhibited reduced severity, shorter duration, and delayed onset. In the KA-SE model, cemdomespib significantly mitigated the severity of SE and reduced the emergence of spontaneous recurrent seizures during the chronic phase, as evidenced by lower seizure frequency, decreased cumulative seizure burden, and prolonged latency to seizure onset. Furthermore, treated animals demonstrated improved anxiety-like behavior and enhanced exploratory activity. SIGNIFICANCE:Cemdomespib confers both acute seizure protection and long-term suppression of epileptogenesis, likely through Hsp90-dependent regulation of mitochondrial integrity and redox signaling. These findings highlight Hsp90 inhibition as a promising therapeutic strategy for seizure control while also mitigating the progression of epileptogenesis and its associated neurobehavioral impairments.

Epilepsy affects 1-2% of the world population, is enigmatic in 30% of cases, and is often intractable, unresponsive to antiepileptic drugs, and accompanied by cognitive, psychiatric and behavioral problems. Tests for Autoimmune Epilepsy are not performed routinely, and limited to passive diagnosis of known autoimmune antibodies, without essential functional tests to reveal active pathogenic antibodies. We investigated two young Epilepsy patients with different Epilepsy characteristics, repeated intractable seizures, and enigmatic etiology. We suspected Autoimmune Epilepsy. We found that both patients have elevated IgG antibodies, and three types of glutamate receptor antibodies, to: AMPA-GluR3B, NMDA-NR1 and NMDA-NR2 peptides. In contrast, they lack autoantibodies to: LGI1, CASPR2, GABA-RB1, Amphiphysin, CV2, PNMA1, Ri, Yo, Hu, Recoverin, Soxi and Titin. IgG antibodies of both patients bound and killed human neural cells In vitro. Moreover, In vivo video EEG studies in naive rats revealed that patient's IgG antibodies, infused continually into rat brain, bound neural cells in the hippocampus and cortex, caused neural loss in these brain regions, and induced recurrent Generalized Tonic Clonic Seizures. We assume they can do so also in the patient's brain. This is the first model of human Autoimmune Epilepsy in rats. It can serve for discovery of patient's pathogenic antibodies, and drug development. Tests for autoimmune antibodies that bind glutamate receptor peptides, and functional diagnostic tests, are obligatory in all enigmatic intractable Epilepsy patients. Current diagnosis of Autoimmune Epilepsy is insufficient! If pathogenic antibodies are found, intractable patients must receive available, suitable and potentially life-changing immunotherapies for Autoimmune Epilepsy.

Epilepsy, a chronic neurological disorder affecting approximately 1 % of the global population, is characterized by recurrent seizures that are often refractory to current antiseizure medications (ASMs). These pharmacotherapies predominantly suppress symptoms without intervening in the underlying pathophysiological cascade, which includes persistent oxidative stress and neuroinflammation, key drivers of epileptogenesis and pharmacoresistance. Among the primary enzymatic sources of reactive oxygen species (ROS), NADPH oxidase 2 (NOX2) has emerged as a central mediator of redox imbalance and neuroimmune activation in the brain. However, the sex-specific roles of NOX2 and its modulation as a therapeutic strategy remain largely unexplored. Here, we investigated the therapeutic efficacy of GSK2795039, a selective and functionally active NOX2 inhibitor, in a kainic acid (KA)-induced status epilepticus (SE) rat model. We examined both acute and chronic outcomes of early NOX2 inhibition on oxidative stress, neuroinflammation, hippocampal neurodegeneration, and cognitive function, incorporating rigorous analysis of sex-dependent responses. Long-term effects on epileptogenesis were assessed using continuous 24/7 video-electrocorticographic (vECoG) monitoring. Our results revealed that early GSK2795039 intervention significantly attenuated SE-induced oxidative damage, pro-inflammatory cytokine expression, and neuronal death, thereby mitigating the development of spontaneous recurrent seizures. Notably, male rats exhibited a more robust therapeutic response, including a marked reduction in seizure burden and improved cognitive performance, whereas females displayed a more modest response, suggesting the presence of compensatory or NOX2-independent antioxidant mechanisms. These findings underscore the pivotal role of NOX2-derived ROS in driving epileptogenesis and highlight the translational potential of NOX2-targeted therapies. Importantly, our study revealed a clear sex divergence in therapeutic outcomes, reinforcing the necessity of integrating sex as a critical biological variable in preclinical and clinical strategies aimed at disease modification in epilepsy.

Oxidative stress is a pivotal driver of epileptogenesis and seizure-induced neuronal pathology, with NADPH oxidase 2 (NOX2) serving as a major source of reactive oxygen species (ROS) in the brain. Despite its established role in seizure pathophysiology, the therapeutic implications of selective NOX2 inhibition in epilepsy remain insufficiently explored. Here, we investigate the effect of GSK2795039, a potent NOX2 inhibitor, using both in vitro and in vivo epilepsy models. In vitro, mixed cortical neuroglial cultures were treated with 4-aminopyridine (4-AP) and picrotoxin (PTX) to induce epileptiform activity. Calcium imaging and dihydroethidium (DHE) fluorescence assays revealed that GSK2795039 significantly reduced synchronous Ca2+ oscillations and ROS accumulation. In vivo, adult rats implanted with ECoG transmitters were pretreated with GSK2795039 prior to pentylenetetrazol (PTZ) administration to evoke seizures. ECoG recording and behavioral seizure scoring showed that GSK2795039 pretreatment inhibited the seizure severity, duration and cumulative seizure burden. Molecular analyses, including quantitative PCR and western blotting, revealed a significant downregulation of NOX2 mRNA in both the hippocampus and cortex, although protein levels remained unchanged. Additionally, immunofluorescence and histological staining confirmed that GSK2795039 mitigated oxidative DNA damage, preserved hippocampal neuronal integrity, and differentially modulated pro- and anti-inflammatory cytokine expression. These findings underscore NOX2 inhibition as a compelling neuroprotective strategy and highlight the potential of GSK2795039 to suppress oxidative and inflammatory cascades in epilepsy. Targeting NOX2 may represent a promising avenue for precision therapeutics in oxidative stress-driven epilepsy.

>Epilepsy is a prevalent chronic brain disorder that is characterized by a persistent predisposition to recurrently generate epileptic seizures and is often associated with cognitive and psychological consequences.Epilepsy affects approximately65 million individuals,including both males and females of all ages worldwide,

Background Epilepsy affects over 65 million people worldwide and significantly burdens patients, caregivers, and society. Drug-resistant epilepsy occurs in approximately 30% of patients and growing evidence indicates that oxidative stress contributes to the development of such epilepsies. Activation of the Nrf2 pathway, which is involved in cellular defense, offers a potential strategy for reducing oxidative stress and epilepsy treatment. Dimethyl fumarate (DMF), an Nrf2 activator, exhibits antioxidant and anti-inflammatory effects and is used to treat multiple sclerosis. Methods The expression of Nrf2 and its related genes in vehicle or DMF treated rats were determined via RT-PCR and Western blot analysis. Neuronal cell death was evaluated by immunohistochemical staining. The effects of DMF in preventing the onset of epilepsy and modifying the disease were investigated in the kainic acid-induced status epilepticus model of temporal lobe epilepsy in rats. The open field, elevated plus maze and T-Maze spontaneous alteration tests were used for behavioral assessments. Results We demonstrate that administration of DMF following status epilepticus increased Nrf2 activity, attenuated status epilepticus-induced neuronal cell death, and decreased seizure frequency and the total number of seizures compared to vehicle-treated animals. Moreover, DMF treatment reversed epilepsy-induced behavioral deficits in the treated rats. Moreover, DMF treatment even when initiated well after the diagnosis of epilepsy, reduced symptomatic seizures long after the drug was eliminated from the body. Conclusions Taken together, these findings suggest that DMF, through the activation of Nrf2, has the potential to serve as a therapeutic target for preventing epileptogenesis and modifying epilepsy.

Background Drug resistance is a particular problem in patients with temporal lobe epilepsy, where seizures originate mainly from the hippocampus. Many of these epilepsies are acquired conditions following an insult to the brain such as a prolonged seizure. Such conditions are characterized by pathophysiological mechanisms including massive oxidative stress that synergistically mediate the secondary brain damage, contributing to the development of epilepsy. The transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) has emerged in recent years as an attractive therapeutic approach targeting to upregulate the antioxidative defenses in the cell, to ameliorate the oxidative stress-induced damage. Thus, it is important to understand the characteristics of Nrf2 activation during epileptogenesis and epilepsy. Here, we studied the temporal, regional, and cell-type specific expression of Nrf2 in the brain, in a rat model of temporal lobe epilepsy. Results Early after status-epilepticus, Nrf2 is mainly activated in the hippocampus and maintained during the whole period of epileptogenesis. Only transient expression of Nrf2 was observed in the cortex. Nevertheless, the expression of several Nrf2 antioxidant target genes was increased within 24 h after status-epilepticus in both the cortex and the hippocampus. We demonstrated that after status-epilepticus in rats, Nrf2 is predominantly expressed in neurons in the CA1 and CA3 regions of the hippocampus, and only astrocytes in the CA1 increase their Nrf2 expression. Conclusions In conclusion, our data identify previously unrecognized spatial and cell-type dependent activation of Nrf2 during epilepsy development, highlighting the need for a time-controlled, and cell-type specific activation of the Nrf2 pathway for mediating anti-oxidant response after brain insult, to modify the development of epilepsy.

The modulation of the nuclear factor erythroid 2-like 2 (Nrf2) activity has been reported to be implicated in the pathology of various neurological disorders, including epilepsy. Previous studies have demonstrated that Nrf2 is activated in the post-status epilepticus rat model; however, the spatiotemporal as well as cell type-specific expression of Nrf2 following brief epileptic seizures remains unclear. Here, we evaluated how an acute epileptic seizure affected the expression of Nrf2 and its downstream genes in the rats' cortex and the hippocampus up to 1 week following the induced seizure. We found that after a pentylenetetrazol-induced seizure, Nrf2 significantly increased at 24 h at the mRNA level and 3 h at the protein level in the cortex. In the hippocampus, the Nrf2 mRNA level peaked at 3 h after the seizure, and no significant changes were observed in the protein level. Interestingly, the mRNA level of Nrf2 downstream genes peaked at 3-6 h after seizure in both the cortex and the hippocampus. A significant increase in the expression of Nrf2 was observed in the neuronal population of CA1 and CA3 regions of the hippocampus, as well as in the cortex. Moreover, we observed no change in the co-localization of Nrf2 with astrocytes neither in the cortex nor in CA1 and CA3. Our results revealed that following a brief acute epileptic seizure, the expression of Nrf2 and its downstream genes is transiently increased and peaked at early timepoints after the seizure predominantly in the hippocampus, and this expression is restricted to the neuronal population.

Recent work by us and others has implicated NADPH oxidase (NOX) enzymes as main producers of reactive oxygen species (ROS) following a brain insult such as status epilepticus, contributing to neuronal damage and development of epilepsy. Although several NOX isoforms have been examined in the context of epilepsy, most attention has focused on NOX2. In this present study, we demonstrate the effect of gp91ds-tat, a specific competitive inhibitor of NOX2, in in vitro epileptiform activity model as well as in temporal lobe epilepsy (TLE) model in rats. We showed that in in vitro seizure model, gp91ds-tat modulated Ca2+ oscillation, prevented epileptiform activity-induced ROS generation, mitochondrial depolarization, and neuronal death. Administration of gp91ds-tat 1 h after kainic acid-induced status epilepticus significantly decreased the expression of NOX2, as well as the overall NOX activity in the cortex and the hippocampus. Finally, we showed that upon continuous intracerebroventricular administration to epileptic rats, gp91ds-tat significantly reduced the seizure frequency and the total number of seizures post-treatment compared to the scrambled peptide-treated animals. The results of the study suggest that NOX2 may have an important effect on modulation of epileptiform activity and has a critical role in mediating seizure-induced NOX activation, ROS generation and oxidative stress in the brain, and thus significantly contributes to development of epilepsy following a brain insult.

The modulation of Nrf2 activity has been reported to be implicated in the pathology of various neurological disorders, including epilepsy. Previous studies have demonstrated that Nrf2 is activated in the post-status epilepticus rat model, however, the spatio-temporal, as well as cell type-specific expression of Nrf2 following brief epileptic seizures remains unclear. Here, we evaluated how an acute epileptic seizure affected the expression of Nrf2 and its downstream genes in the cortex and the hippocampus up to 1-week following the induced seizure. We found that after a pentylenetetrazol-induced seizure, Nrf2 significantly increased at 24 h at the mRNA level and 3 to 6 h at the protein level in the cortex. In the hippocampus, the Nrf2 mRNA level peaked at 3 h after the seizure, and no significant changes were observed in the protein level. Interestingly, the mRNA level of Nrf2 downstream genes peaked at 3-6 h after seizure in both the cortex and the hippocampus. A significant increase in the expression of Nrf2 was observed in the neuronal population of CA1 and CA3 regions of the hippocampus, as well as in the cortex. Moreover, we observed no change in the co-localization of Nrf2 with astrocytes neither in the cortex nor in CA1 and CA3. Our results revealed that following a brief acute epileptic seizure, the expression of Nrf2 and its downstream genes is transiently increased and peaked at early timepoints after seizure predominantly in the hippocampus, and this expression is restricted to the neuronal population.

Diabetes is a group of metabolic disorders characterized by elevated blood sugar levels, leading to many undesirable health consequences. There are many herbal formulations, traditionally used by the Northeast Indian population for disease management. These formulations require scientific validations to optimize their efficacy and increase their popularity. In this study, we attempt to scientifically validate a polyherbal formulation traditionally used for the management of diabetes through preliminary phytochemicals investigation, characterization of potential phytochemicals using Fourier transform infrared (FT-IR) spectroscopy, high-resolution liquid chromatography mass spectrometry (HR-LC/MS) analysis, and in silico characterization of physiochemical, drug-likeness, and pharmacokinetic properties of identified phytochemical compounds. Qualitative phytochemical screening of various extracts of the formulation confirmed the presence of alkaloids, phenols and tannins, flavonoids, fats, and oils. Phytochemical quantification of the various extracts showed that the highest total phenolic content is present in the ethanolic extract (35.61 ± 0.15 mg GAE/g), while the highest total flavonoid content is present in the chloroform extract (76.33 ± 2.96 mg QE/g) of the formulation. FT-IR spectroscopic analysis revealed various characteristic band values with various functional groups in the formulation extract such as amines, alcohol, fluoro compounds, phenol, alkane, alkene, and conjugated acid groups. HR-LC/MS analyses identified nearly 51 compounds including 9 small peptides and 42 potential phytochemical compounds. In silico SwissADME analysis of identified compounds revealed 25 potential compounds following Lipinski's rule and showing drug-like characteristics, and out of them, 16 compounds exhibited good oral bioavailability, as revealed in the bioavailability radar. The overall study showed that the presented polyherbal formulation is enriched with bio-active phytochemical compounds with good pharmaceutical values.

The NADPH Oxidase (NOX) enzymes are key producers of reactive oxygen species (ROS) and consist of seven different isoforms, distributed across the tissues and cell types. The increasing level of ROS induces oxidative stress playing a crucial role in neuronal death and the development of epilepsy. Recently, NOX2 was reported as a primary source of ROS production, activated by NMDA receptor, a crucial marker of epilepsy development. Here, we demonstrate spatial, temporal, and cellular expression of NOX2 and NOX4 complexes in in-vitro and in-vivo seizure models. We showed that the expression of NOX2 and NOX4 was increased in the initial 24 h following a brief seizure induced by pentylenetetrazol. Interestingly, while this elevated level returns to baseline 48 h following seizure in the cortex, in the hippocampus these levels remain elevated up to one week following the seizure. Moreover, we showed that 1- and 2- weeks following status epilepticus (SE), expression of NOX2 and NOX4 remains significantly elevated both in the cortex and the hippocampus. Furthermore, in in-vitro seizure model, NOX2 and NOX4 isoforms were overexpressed in neurons and astrocytes following seizures. These results suggest that NOX2 and NOX4 in the brain have a transient response to seizures, and these responses temporally vary depending on, seizure duration, brain region (cortex or hippocampus), and cell types.

Background: The mechanistic (or mammalian) target of rapamycin (mTOR), a Ser/Thr kinase, associates with different subunits forming two functionally distinct complexes, mTORC1 and mTORC2, regulating a diverse set of cellular functions in response to growth factors, cellular energy levels, and nutrients. The mechanisms regulating mTORC1 activity are well characterized; regulation of mTORC2 activity, however, remains obscure. While studies conducted in Dictyostelium suggest a possible role of Ras protein as a potential upstream regulator of mTORC2, definitive studies delineating the underlying molecular mechanisms, particularly in mammalian cells, are still lacking. Methods: Protein levels were measured by Western blotting and kinase activity of mTORC2 was analyzed by in vitro kinase assay. In situ Proximity ligation assay (PLA) and co-immunoprecipitation assay was performed to detect protein-protein interaction. Protein localization was investigated by immunofluorescence and subcellular fractionation while cellular function of mTORC2 was assessed by assaying extent of cell migration and invasion. Results: Here, we present experimental evidence in support of the role of Ras activation as an upstream regulatory switch governing mTORC2 signaling in mammalian cancer cells. We report that active Ras through its interaction with mSIN1 accounts for mTORC2 activation, while disruption of this interaction by genetic means or via peptide-based competitive hindrance, impedes mTORC2 signaling. Conclusions: Our study defines the regulatory role played by Ras during mTORC2 signaling in mammalian cells and highlights the importance of Ras-mSIN1 interaction in the assembly of functionally intact mTORC2.