Neuronal Activity Promotes Glioma Progression by Inducing Proneural-to-Mesenchymal Transition in Glioma Stem Cells.

Neuronal activity can drive progression of high-grade glioma by mediating mitogen production and neuron-glioma synaptic communications. Glioma stem cells (GSC) also play a significant role in progression, therapy resistance, and recurrence in glioma, which implicates potential cross-talk between neuronal activity and GSC biology. Here, we manipulated neuronal activity using chemogenetics in vitro and in vivo to study how it influences GSCs. Neuronal activity supported glioblastoma (GBM) progression and radioresistance through exosome-induced proneural-to-mesenchymal transition (PMT) of GSCs. Molecularly, neuronal activation led to elevated miR-184-3p in neuron-derived exosomes that were taken up by GSCs and reduced the mRNA N6-methyladenosine (m6A) levels by inhibiting RBM15 expression. RBM15 deficiency decreased m6A modification of DLG3 mRNA and subsequently induced GSC PMT by activating the STAT3 pathway. Loss of miR-184-3p in cortical neurons reduced GSC xenograft growth, even when neurons were activated. Levetiracetam, an antiepileptic drug, reduced the neuronal production of miR-184-3p-enriched exosomes, inhibited GSC PMT, and increased radiosensitivity of tumors to prolong survival in xenograft mouse models. Together, these findings indicate that exosomes derived from active neurons promote GBM progression and radioresistance by inducing PMT of GSCs. SIGNIFICANCE: Active neurons secrete exosomes enriched with miR-184-3p that promote glioblastoma progression and radioresistance by driving the proneural-to-mesenchymal transition in glioma stem cells, which can be reversed by antiseizure medication levetiracetam.

Hypoxia-Induced Neuronal Activity in Glioma Patients Polarizes Microglia by Potentiating RNA m6A Demethylation.

PURPOSE: Neuronal activity in the brain has been reported to promote the malignant progression of glioma cells via nonsynaptic paracrine and electrical synaptic integration mechanisms. However, the interaction between neuronal activity and the immune microenvironment in glioblastoma (GBM) remains largely unclear. EXPERIMENTAL DESIGN: By applying chemogenetic techniques, we enhanced and inhibited neuronal activity in vitro and in a mouse model to study how neuronal activity regulates microglial polarization and affects GBM progression. RESULTS: We demonstrate that hypoxia drove glioma stem cells (GSC) to produce higher levels of glutamate, which activated local neurons. Neuronal activity promoted GBM progression by facilitating microglial M2 polarization through enriching miR-200c-3p in neuron-derived exosomes, which decreased the expression of the m6A writer zinc finger CCCH-type containing 13 (ZC3H13) in microglia, impairing methylation of dual specificity phosphatase 9 (DUSP9) mRNA. Downregulation of DUSP9 promoted ERK pathway activation, which subsequently induced microglial M2 polarization. In the mouse model, cortical neuronal activation promoted microglial M2 polarization whereas cortical neuronal inhibition decreased microglial M2 polarization in GBM xenografts. miR-200c-3p knockdown in cortical neurons impaired microglial M2 polarization and GBM xenograft growth, even when cortical neurons were activated. Treatment with the anti-seizure medication levetiracetam impaired neuronal activation and subsequently reduced neuron-mediated microglial M2 polarization. CONCLUSIONS: These findings indicated that hypoxic GSC-induced neuron activation promotes GBM progression by polarizing microglia via the exosomal miR-200c-3p/ZC3H13/DUSP9/p-ERK pathway. Levetiracetam, an antiepileptic drug, blocks the abnormal activation of neurons in GBM and impairs activity-dependent GBM progression. See related commentary by Cui et al., p. 1073.

Apolipoprotein E3 functionalized lipid-drug conjugated nanoparticles of Levetiracetam for enhanced delivery to the brain: In-vitro cell line studies and in-vivo study.

A significant portion of brain-tumor patients suffer from 'brain-tumor-related epilepsy (BTE)' which results in depression, anxiety and hampered quality of life. Conventional anti-epileptic drugs indicate negative interaction with other drugs augmenting the poor outcome of overall therapy. Levetiracetam (LVM) has evidenced effectiveness for BTE but its hydrophilicity restricts the passage into blood-brain barrier. The majority of lipid nanoparticles fails to load hydrophilic drug sufficiently. Therefore, lipid-drug conjugates (LDC) were synthesized using stearic acid via amide bond formation confirmed by FTIR and NMR. The nanoparticles of synthesized LDC were prepared by solvent injection method followed by functionalization with Apolipoprotein E3 (ApoE3@LDC-NP). The nanoparticles were characterized by DSC, XRD, particle size (131.6 ± 1.24 nm), zeta potential (-15.6 ± 0.09 mV), and for storage stability. In-vitro release study indicated initial burst release of 20 ± 0.63 % followed by sustained release up to 30 h (66 ± 1.40 %) for ApoE3@LDC-NP. The cell-line study on HEK293 indicated no significant cytotoxic effect and greater cell uptake through U87MG cell line. The pharmacokinetic and bio-distribution study indicated 2.5-fold greater brain-targeting of ApoE3@LDC-NP as compared to LVM solution. It proved safe in the haemolysis study and exhibited the absence of tissue necrosis. Thus, ApoE3@LDC-NP might be a promising approach for effective brain-targeting of LVM for improved clinical response in BTE.

Assessment of the toxic effects of levetiracetam on biochemical, functional, and redox parameters of salivary glands in male Wistar rats.

Levetiracetam (LEV) is an anticonvulsant for epilepsy. The toxic effects of this medication in tissues have been associated with redox state imbalance, which can lead to salivary gland dysfunction. Therefore, the current work investigated the effects of LEV on the biochemical, functional, and redox parameters of the parotid and submandibular glands in rats. For this, male Wistar rats (Rattus norvegicus albinus) were randomly divided into 3 groups (n = 10/group): Control (0.9% saline solution), LEV100 (100 mg/kg), and LEV300 (300 mg/kg). After 21 consecutive days of intragastric gavage treatments, pilocarpine stimulated saliva secretion was collected for salivary biochemical analysis. The extracted salivary glands were utilized for histomorphometry and redox state analyses. Our results showed that LEV300 increased plasma hepatotoxicity markers and reduced salivary amylase activity and the acinar surface area of the parotid gland. Total oxidant capacity and oxidative damage to lipids and proteins were higher in the parotid gland, while total antioxidant capacity and uric acid levels were reduced in the submandibular gland of the LEV100 group compared to Control. On the other hand, total oxidant capacity, oxidative damage to lipids and proteins, total antioxidant capacity, and uric acid levels were lower in both salivary glands of the LEV300 group compared to Control. Superoxide dismutase and glutathione peroxidase activities were lower in the salivary glands of treated animals compared to Control. In conclusion our data suggest that treatment with LEV represents a potentially toxic agent, that contributes to drug-induced salivary gland dysfunction.

Quercetin protects against levetiracetam induced gonadotoxicity in rats.

The purpose of this study was to determine whether quercetin may counteract the negative effects of levetiracetam on rat reproductive capabilities by examining its influence on a few reproductive parameters following levetiracetam administration. Twenty (20) experimental rats were employed, with five (n = 5) animals per treatment group. Rats in group 1 received saline (10 mL/kg, p.o.) which served as control. Quercetin (20 mg/kg, p.o./day) was given to groups 2 and 4 for 28 days starting from 29 to 56 days, respectively. However, animals in groups 3-4 received LEV (300 mg/kg) once daily for 56 days with a 30-minute break in between treatments. All rats had their serum sex hormone levels, sperm characteristics, testicular antioxidant capability, and levels of oxido-inflammatory/apoptotic mediators evaluated. Additionally, the expression of proteins associated to BTB, autophagy, stress response was examined in rat testes. LEV increased sperm morphological defects and decreased sperm motility, sperm viability, sperm count body weight and testes weight, MDA and 8OHdG levels in the testis of LEV-treated rats were elevated, while antioxidant enzyme expression was concurrently decreased. Additionally, it reduced the levels of serum gonadotropins, testosterone, mitochondrial membrane potential, and cytochrome C liberation into the cytosol from the mitochondria. Caspase-3 and Caspase-9 activity increased. While Bcl-2, Cx-43, Nrf2, HO-1, mTOR, and Atg-7 levels were lowered, NOX-1, TNF-α, NF-kß, IL-1ß, and tDFI levels increased. Histopathological scoring provided further support for the decreased spermatogenesis. In contrast to all of these gonadotoxic effects of LEV, improvements in LEV-induced gonadal damage were seen through upregulation of Nrf2/ HO-1, Cx-43/NOX-1, mTOR/Atg-7 expression and attenuation of hypogonadism, poor sperm quality, mitochondria-mediated apoptosis, and oxidative inflammation due to quercetin post-treatment. The modulation of Nrf2/HO-1, /mTOR/Atg-7 and Cx-43/NOX-1 levels and the inhibition of mitochondria-mediated apoptosis and oxido-inflammation in LEV-induced gonadotoxicity in rats suggest that quercetin may hold promise as a possible therapeutic treatment.

Acute administration of levetiracetam in tonic pain model modulates gene expression of 5HT1A and 5HT7 receptors in the thalamus of rats (Rattus norvergicus).

The nociceptive effect of Levetiracetam (LEV) on the expression of 5-HT1A and 5-HT7 receptors found in the thalamus was evaluated. Thirty-six male rats (Wistar) were randomized into six groups: in the Control group without treatment; LEV50 group LEV was administered in a single dose of 50 mg/kg i.g.; in the LEV300 group LEV dose of 300 mg/kg i.g.; in the FORMALIN group the formalin test was performed; in the LEV50/FORMALIN group LEV dose of 50 mg/kg i.g and the formalin test was performed; in the LEV300/FORMALIN group LEV dose of 300 mg/kg i.g and the formalin test was performed, subsequently the thalamus was dissected in all groups. In the formalin tests LEV exhibited an antinociceptive effect in the LEV300/FORMALIN group (p < 0.05) and a pronociceptive effect in the LEV50/FORMALIN group (p < 0.001). The results obtained by Real-time PCR confirmed the expression of the 5-HT1A and 5-HT7 receptors in the thalamus, 5-HT1A receptors increased significantly in the FORMALIN group and the LEV300/FORMALIN group (p < 0.05). 5-HT7 receptors are only over expressed at a dose of 300 mg/Kg of LEV with formalin (p < 0.05). This suggests that LEV modulates the sensation of pain by controlling the expression of 5-HT1A and 5-HT7 in a tonic pain model, and that changes in the expression of 5-HT1A and 5-HT7 receptors are associated with the sensation of pain, furthermore its possibility to be used in clinical treatments for pain.

El levetiracetam puede disminuir la dosis de desmopresina en niños con diabetes insípida / Levetiracetam can decrease desmopressin requirement in children with diabetes insipidus

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Levetiracetam Protects Against Cognitive Impairment of Subthreshold Convulsant Discharge Model Rats by Activating Protein Kinase C (PKC)-Growth-Associated Protein 43 (GAP-43)-Calmodulin-Dependent Protein Kinase (CaMK) Signal Transduction Pathway.

BACKGROUND Subclinical epileptiform discharges (SEDs) are defined as epileptiform electroencephalographic (EEG) discharges without clinical signs of seizure in patients. The subthreshold convulsant discharge (SCD) is a frequently used model for SEDs. This study aimed to investigate the effect of levetiracetam (LEV), an anti-convulsant drug, on cognitive impairment of SCD model rats and to assess the associated mechanisms. MATERIAL AND METHODS A SCD rat model was established. Rats were divided into an SCD group, an SCD+ sodium valproate (VPA) group, and an SCD+ levetiracetam (LEV) group. The Morris water maze was used to evaluate the capacity of positioning navigation and space exploration. The field excitatory post-synaptic potentials (fEPSPs) were evaluated using a bipolar stimulation electrode. NCAM, GAP43, PS95, and CaMK II levels were detected using Western blot and RT-PCR, respectively. PKC activity was examined by a non-radioactive method. RESULTS LEV shortens the latency of platform seeking in SCD rats in positioning navigation. fEPSP slopes were significantly lower in the SCD group, and LEV treatment significantly enhanced the fEPSP slopes compared to the SCD group (P<0.05). The NCAM and GAP-43 levels were increased and PSD-95 levels were increased in SCD rats (P<0.05), which were improved by LEV treatment. The PKC activity and CaMK II levels were decreased in SCD rats and LEV treatment significantly enhanced PKC activity and increased CaMK II levels. CONCLUSIONS Cognitive impairment in of SCD model rats may be caused by decreased PKC activity, low expression of CaMK II, and inhibition of LTP formation. LEV can improve cognitive function by activating the PKC-GAP-43-CaMK signal transduction pathway.

Effects of Three Anti-Seizure Drugs on Cholinergic and Metabolic Activity in Experimental Status Epilepticus.

PURPOSE: Status epilepticus (SE) is characterized by recurrent seizure activity and can be drug- resistant. Knowledge of neuronal and metabolic activity of the brain during SE may be helpful to improve medical care. We here report the effects of three anti-seizure drugs on changes of acetylcholine energy metabolites and oxidative stress during SE. METHODS: We used the lithium-pilocarpine model in rats to induce SE and in vivo- microdialysis to monitor cholinergic and metabolic activity in the hippocampus. We measured extracellular concentrations of acetylcholine, glucose, lactate, pyruvate, glycerol and isoprostanes before and during SE, and after acute treatment with pregabalin, valproic acid, and levetiracteam. RESULTS: Upon onset of  SE, acetylcholine (ACh) release increased six- to eightfold. Glucose was increased only transiently by 30% but lactate levels rose four-fold, and extracellular concentrations of glycerol ten-fold. Isoprostanes are markers of oxidative stress and increased more than 20-fold. Two hours after pilocarpine adminstration, rats were treated with pregabalin (100 mg/kg), levetiracetam (200 mg/kg) or valproic acid (400 mg/kg) by i.p. injection. All three drugs stopped seizure activity in a delayed fashion, but at the doses indicated, only animals that received levetiracetam reached consciousness. All drugs reduced ACh release within 60-120 minutes. Lactate/pyruvate ratios, glycerol and isoprostanne levels were also reduced significantly after drug administration. CONCLUSIONS: Hippocampal ACh release closely follows seizure activity in SE and is attenuated when SE subsides. Pregabalin, valproic acid and levetiracetam all terminate seizures in the rat SE model and attenuate cholinergic and metabolic changes within two hours.

A single-center, open-label positron emission tomography study to evaluate brivaracetam and levetiracetam synaptic vesicle glycoprotein 2A binding in healthy volunteers.

OBJECTIVE: Brivaracetam (BRV) and levetiracetam (LEV) are antiepileptic drugs that bind synaptic vesicle glycoprotein 2A (SV2A). In vitro and in vivo animal studies suggest faster brain penetration and SV2A occupancy (SO) after dosing with BRV than LEV. We evaluated human brain penetration and SO time course of BRV and LEV at therapeutically relevant doses using the SV2A positron emission tomography (PET) tracer 11 C-UCB-J (EP0074; NCT02602860). METHODS: Healthy volunteers were recruited into three cohorts. Cohort 1 (n = 4) was examined with PET at baseline and during displacement after intravenous BRV (100 mg) or LEV (1500 mg). Cohort 2 (n = 5) was studied during displacement and 4 hours postdose (BRV 50-200 mg or LEV 1500 mg). Cohort 3 (n = 4) was examined at baseline and steady state after 4 days of twice-daily oral dosing of BRV (50-100 mg) and 4 hours postdose of LEV (250-600 mg). Half-time of 11 C-UCB-J signal change was computed from displacement measurements. Half-saturation concentrations (IC50 ) were determined from calculated SO. RESULTS: Observed tracer displacement half-times were 18 ± 6 minutes for BRV (100 mg, n = 4), 9.7 and 10.1 minutes for BRV (200 mg, n = 2), and 28 ± 6 minutes for LEV (1500 mg, n = 6). Estimated corrected half-times were 8 minutes shorter. The SO was 66%-70% for 100 mg intravenous BRV, 84%-85% for 200 mg intravenous BRV, and 78%-84% for intravenous 1500 mg LEV. The IC50 of BRV (0.46 µg/mL) was 8.7-fold lower than of LEV (4.02 µg/mL). BRV data fitted a single SO versus plasma concentration relationship. Steady state SO for 100 mg BRV was 86%-87% (peak) and 76%-82% (trough). SIGNIFICANCE: BRV achieves high SO more rapidly than LEV when intravenously administered at therapeutic doses. Thus, BRV may have utility in treating acute seizures; further clinical studies are needed for confirmation.