Novel predictive approaches for drug-induced convulsions in non-human primates using machine learning and heart rate variability analysis.

Drug-induced convulsions are a major challenge to drug development because of the lack of reliable biomarkers. Using machine learning, our previous research indicated the potential use of an index derived from heart rate variability (HRV) analysis in non-human primates as a biomarker for convulsions induced by GABAA receptor antagonists. The present study aimed to explore the application of this methodology to other convulsants and evaluate its specificity by testing non-convulsants that affect the autonomic nervous system. Telemetry-implanted males were administered various convulsants (4-aminopyridine, bupropion, kainic acid, and ranolazine) at different doses. Electrocardiogram data gathered during the pre-dose period were employed as training data, and the convulsive potential was evaluated using HRV and multivariate statistical process control. Our findings show that the Q-statistic-derived convulsive index for 4-aminopyridine increased at doses lower than that of the convulsive dose. Increases were also observed for kainic acid and ranolazine at convulsive doses, whereas bupropion did not change the index up to the highest dose (1/3 of the convulsive dose). When the same analysis was applied to non-convulsants (atropine, atenolol, and clonidine), an increase in the index was noted. Thus, the index elevation appeared to correlate with or even predict alterations in autonomic nerve activity indices, implying that this method might be regarded as a sensitive index to fluctuations within the autonomic nervous system. Despite potential false positives, this methodology offers valuable insights into predicting drug-induced convulsions when the pharmacological profile is used to carefully choose a compound.

Chemical and biophysical characterization of novel potassium channel blocker 3-fluoro-5-methylpyridin-4-amine.

4-aminopyridine (4AP) is a potassium (K+) channel blocker used clinically to improve walking in people with multiple sclerosis (MS). 4AP binds to exposed K+ channels in demyelinated axons, reducing the leakage of intracellular K+ and enhancing impulse conduction. Multiple derivatives of 4AP capable of blocking K+ channels have been reported including three radiolabeled with positron emitting isotopes for imaging demyelinated lesions using positron emission tomography (PET). However, there remains a demand for novel molecules with suitable physicochemical properties and binding affinity that can potentially be radiolabeled and used as PET radiotracers. In this study, we introduce 3-fluoro-5-methylpyridin-4-amine (5Me3F4AP) as a novel trisubstituted K+ channel blocker with potential application in PET. 5Me3F4AP has comparable potency to 4AP and the PET tracer 3-fluoro-4-aminopyridine (3F4AP). Compared to 3F4AP, 5Me3F4AP exhibits comparable basicity (pKa = 7.46 ± 0.01 vs. 7.37 ± 0.07, P-value = 0.08), greater lipophilicity (logD = 0.664 ± 0.005 vs. 0.414 ± 0.002, P-value < 0.0001) and higher permeability to an artificial brain membrane (Pe = 88.1 ± 18.3 vs. 31.1 ± 2.9 nm/s, P-value = 0.03). 5Me3F4AP is also more stable towards oxidation in vitro by the cytochrome P450 enzyme CYP2E1 (IC50 = 36.2 ± 2.5 vs. 15.4 ± 5.1, P-value = 0.0003); the enzyme responsible for the metabolism of 4AP and 3F4AP. Taken together, 5Me3F4AP has promising properties as a candidate for PET imaging warranting additional investigation.

Alarin potentiates ongoing epileptiform activity in rat brain slices: an in vitro electrophysiological study.

Alarin is a newly discovered neuropeptide that belongs to the galanin peptide family with a wide range of bioactivity in the nervous system. Its function in the brain's autonomic areas has been studied, and it has been reported that alarin is involved in the regulation of excitability in hypothalamic neurons. Its role in the regulation of excitability in the hippocampus, however, is unknown. In this study, we investigated if alarin induced any synchronous discharges or epileptiform activity, and if it had any effect on already initiated epileptiform discharges. We used thick acute horizontal hippocampal slices obtained from 30­ to 35­day­old rats. Extracellular field potential recordings were evaluated in the CA1 region of the hippocampus. Our data demonstrated that, alarin application did not result in any epileptiform activity or abnormal discharges. 4­aminopyridine was applied to induce epileptiform activity in the slices. We found that alarin increased the frequency of interictal­like events and the mean power of local field potentials in the CA1 region of the hippocampus, which was induced by 4­aminopyridine. These results demonstrated for the first time that alarin has a modulatory effect on synchronized neuronal discharges and showed the contribution of the neuropeptide alarin to epilepsy­like conditions.

[Effect of asiaticoside on systolic blood pressure and relaxation of isolated thoracic aorta of rats].

OBJECTIVE: To investigate the effect of asiaticoside on blood pressure and relaxation of thoracic aorta in rats and explore the underlying mechanism. METHODS: SD rats treated with 50 and 100 mg/kg asiaticoside by daily gavage for 2 weeks were monitored for systolic blood pressure changes, and histological changes of the thoracic aorta were evaluated using HE staining. In isolated rat endothelium-intact and endothelium-denuded thoracic aorta rings, the effects of asiaticoside on relaxation of the aortic rings were tested at baseline and following norepinephrine (NE)- and KCl-induced constriction. The vascular relaxation effect of asiaticoside was further observed in NE-stimulated endothelium-intact rat aortic rings pretreated with L-nitroarginine methyl ester, indomethacin, zinc protoporphyrin Ⅸ, tetraethyl ammonium chloride, glibenclamide, barium chloride, Iberiotoxin, 4-aminopyridine, or TASK-1-IN-1. The aortic rings were treated with KCl and NE followed by increasing concentrations of CaCl2 to investigate the effect of asiaticoside on vasoconstriction induced by external calcium influx and internal calcium release. RESULTS: Asiaticoside at 50 and 100 mg/kg significantly lowered systolic blood pressure in rats without affecting the thoracic aorta histomorphology. While not obviously affecting resting aortic rings with intact endothelium, asiaticoside at 100 mg/kg induced significant relaxation of the rings constricted by KCl and NE, but its effects differed between endothelium-intact and endothelium-denuded rings. In endothelium-intact aortic rings pretreated with indomethacin, ZnPP Ⅸ, barium chloride, glyburide, TASK-1-IN-1 and 4-aminopyridine, asiaticoside did not produce significant effect on NE-induced vasoconstriction, and tetraethylammonium, Iberiotoxin and L-nitroarginine methyl ester all inhibited the relaxation effect of asiaticoside. In KCland NE-treated rings, asiaticoside obviously inhibited CaCl2-induced vascular contraction. CONCLUSION: Asiaticoside induces thoracic aorta relaxation by mediating high-conductance calcium-activated potassium channel opening, promoting nitric oxide release from endothelial cells and regulating Ca2+ influx and outflow, thereby reducing systolic blood pressure in rats.

RNA Sequencing Demonstrates Ex Vivo Neocortical Transcriptomic Changes Induced by Epileptiform Activity in Male and Female Mice.

Seizures are generally associated with epilepsy but may also be a symptom of many other neurological conditions. A hallmark of a seizure is the intensity of the local neuronal activation, which can drive large-scale gene transcription changes. Such changes in the transcriptional profile likely alter neuronal function, thereby contributing to the pathological process. Therefore, there is a strong clinical imperative to characterize how gene expression is changed by seizure activity. To this end, we developed a simplified ex vivo technique for studying seizure-induced transcriptional changes. We compared the RNA sequencing profile in mouse neocortical tissue with up to 3 h of epileptiform activity induced by 4-aminopyridine (4AP) relative to control brain slices not exposed to the drug. We identified over 100 genes with significantly altered expression after 4AP treatment, including multiple genes involved in MAPK, TNF, and neuroinflammatory signaling pathways, all of which have been linked to epilepsy previously. Notably, the patterns in male and female brain slices were almost identical. Various immediate early genes were among those showing the largest upregulation. The set of down-regulated genes included ones that might be expected either to increase or to decrease neuronal excitability. In summary, we found the seizure-induced transcriptional profile complex, but the changes aligned well with an analysis of published epilepsy-associated genes. We discuss how simple models may provide new angles for investigating seizure-induced transcriptional changes.

GAA-FGF14 disease: defining its frequency, molecular basis, and 4-aminopyridine response in a large downbeat nystagmus cohort.

BACKGROUND: GAA-FGF14 disease/spinocerebellar ataxia 27B is a recently described neurodegenerative disease caused by (GAA)≥250 expansions in the fibroblast growth factor 14 (FGF14) gene, but its phenotypic spectrum, pathogenic threshold, and evidence-based treatability remain to be established. We report on the frequency of FGF14 (GAA)≥250 and (GAA)200-249 expansions in a large cohort of patients with idiopathic downbeat nystagmus (DBN) and their response to 4-aminopyridine. METHODS: Retrospective cohort study of 170 patients with idiopathic DBN, comprising in-depth phenotyping and assessment of 4-aminopyridine treatment response, including re-analysis of placebo-controlled video-oculography treatment response data from a previous randomised double-blind 4-aminopyridine trial. FINDINGS: Frequency of FGF14 (GAA)≥250 expansions was 48% (82/170) in patients with idiopathic DBN. Additional cerebellar ocular motor signs were observed in 100% (82/82) and cerebellar ataxia in 43% (35/82) of patients carrying an FGF14 (GAA)≥250 expansion. FGF14 (GAA)200-249 alleles were enriched in patients with DBN (12%; 20/170) compared to controls (0.87%; 19/2191; OR, 15.20; 95% CI, 7.52-30.80; p < 0.0001). The phenotype of patients carrying a (GAA)200-249 allele closely mirrored that of patients carrying a (GAA)≥250 allele. Patients carrying a (GAA)≥250 or a (GAA)200-249 allele had a significantly greater clinician-reported (80%, 33/41 vs 31%, 5/16; RR, 2.58; 95% CI, 1.23-5.41; Fisher's exact test, p = 0.0011) and self-reported (59%, 32/54 vs 11%, 2/19; RR, 5.63; 95% CI, 1.49-21.27; Fisher's exact test, p = 0.00033) response to 4-aminopyridine treatment compared to patients carrying a (GAA)<200 allele. Placebo-controlled video-oculography data, available for four patients carrying an FGF14 (GAA)≥250 expansion, showed a significant decrease in slow phase velocity of DBN with 4-aminopyridine, but not placebo. INTERPRETATION: This study confirms that FGF14 GAA expansions are a frequent cause of DBN syndromes. It provides preliminary evidence that (GAA)200-249 alleles might be pathogenic. Finally, it provides large real-world and preliminary piloting placebo-controlled evidence for the efficacy of 4-aminopyridine in GAA-FGF14 disease. FUNDING: This work was supported by the Clinician Scientist program "PRECISE.net" funded by the Else Kröner-Fresenius-Stiftung (to CW, AT, and MSy), the grant 779257 "Solve-RD" from the European's Union Horizon 2020 research and innovation program (to MSy), and the grant 01EO 1401 by the German Federal Ministry of Education and Research (BMBF) (to MSt). This work was also supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) N° 441409627, as part of the PROSPAX consortium under the frame of EJP RD, the European Joint Programme on Rare Diseases, under the EJP RD COFUND-EJP N° 825575 (to MSy, BB and-as associated partner-SZ), the NIH National Institute of Neurological Disorders and Stroke (grant 2R01NS072248-11A1 to SZ), the Fondation Groupe Monaco (to BB), and the Montreal General Hospital Foundation (grant PT79418 to BB). The Care4Rare Canada Consortium is funded in part by Genome Canada and the Ontario Genomics Institute (OGI-147 to KMB), the Canadian Institutes of Health Research (CIHR GP1-155867 to KMB), Ontario Research Foundation, Genome Quebec, and the Children's Hospital of Eastern Ontario Foundation. The funders had no role in the conduct of this study.

4-aminopyridine improves evoked potentials and ambulation in the taiep rat: A model of hypomyelination with atrophy of basal ganglia and cerebellum.

The taiep rat is a tubulin mutant with an early hypomyelination followed by progressive demyelination of the central nervous system due to a point mutation in the Tubb4a gene. It shows clinical, radiological, and pathological signs like those of the human leukodystrophy hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC). Taiep rats had tremor, ataxia, immobility episodes, epilepsy, and paralysis; the acronym of these signs given the name to this autosomal recessive trait. The aim of this study was to analyze the characteristics of somatosensory evoked potentials (SSEPs) and motor evoked potentials (MEPs) in adult taiep rats and in a patient suffering from H-ABC. Additionally, we evaluated the effects of 4-aminopyridine (4-AP) on sensory responses and locomotion and finally, we compared myelin loss in the spinal cord of adult taiep and wild type (WT) rats using immunostaining. Our results showed delayed SSEPs in the upper and the absence of them in the lower extremities in a human patient. In taiep rats SSEPs had a delayed second negative evoked responses and were more susceptible to delayed responses with iterative stimulation with respect to WT. MEPs were produced by bipolar stimulation of the primary motor cortex generating a direct wave in WT rats followed by several indirect waves, but taiep rats had fused MEPs. Importantly, taiep SSEPs improved after systemic administration of 4-AP, a potassium channel blocker, and this drug induced an increase in the horizontal displacement measured in a novelty-induced locomotor test. In taiep subjects have a significant decrease in the immunostaining of myelin in the anterior and ventral funiculi of the lumbar spinal cord with respect to WT rats. In conclusion, evoked potentials are useful to evaluate myelin alterations in a leukodystrophy, which improved after systemic administration of 4-AP. Our results have a translational value because our findings have implications in future medical trials for H-ABC patients or with other leukodystrophies.

Progression of axonal excitability abnormalities with increasing clinical severity of diabetic peripheral neuropathy.

OBJECTIVE: Diabetic peripheral neuropathy (DPN) is a frequent complication for persons with type 2 diabetes. Previous studies have failed to demonstrate any significant impact of treatment for DPN. The present study assessed the role of axonal ion channel dysfunction in DPN and explored the hypothesis that there may be a progressive change in ion channel abnormalities that varied with disease stage. METHODS: Neurophysiological studies were conducted using axonal excitability techniques, a clinical method of assessing ion channel dysfunction. Studies were conducted in 178 persons with type 2 diabetes, with participants allocated into four groups according to clinical severity of neuropathy, assessed using the Total Neuropathy Grade. RESULTS: Analysis of excitability data demonstrated a progressive and stepwise reduction in two parameters that are related to the activity of Kv1.1 channels, namely superexcitability and depolarizing threshold electrotonus at 10-20 ms (p < 0.001), and mathematical modelling of axonal excitability findings supported progressive upregulation of Kv1.1 conductances with increasing greater disease severity. CONCLUSION: The findings are consistent with a progressive upregulation of juxtaparanodal Kv1.1 conductances with increasing clinical severity of diabetic peripheral neuropathy. SIGNIFICANCE: From a translational perspective, the study suggests that blockade of Kv1.1 channels using 4-aminopyridine derivatives such as fampridine may be a potential treatment for DPN.

Performance Measures and Plasma Biomarker Levels in Patients with Multiple Sclerosis after 14 Days of Fampridine Treatment: An Explorative Study.

Peripheral cytokine levels may serve as biomarkers for treatment response and disease monitoring in patients with multiple sclerosis (pwMS). The objectives were to assess changes in plasma biomarkers in PwMS after 14 days of fampridine treatment and to explore correlations between changes in performance measures and plasma biomarkers. We included 27 PwMS, 14 women and 13 men, aged 52.0 ± 11.6 years, with a disease duration of 17 ± 8.5 years, and an Expanded Disability Status Scale of 6 [IQR 5.0/6.5]. Gait and hand function were assessed using performance tests completed prior to fampridine and after 14 days of treatment. Venous blood was obtained, and chemiluminescence analysis conducted to assess plasma cytokines and neurodegenerative markers. All performance measures demonstrated improvements. Biomarkers showed decreased tumor necrosis factor (TNF) receptor-2 levels. Associations were found between change scores in (i) Six Spot Step Test and Interleukin (IL)-2, IL-8, and IL-17 levels; (ii) timed 25-foot walk and interferon-γ, IL-2, IL-8, TNF-α, and neurofilament light levels, and (iii) 12-Item Multiple Sclerosis Walking Scale and IL-17 levels. The associations may reflect increased MS-related inflammatory activity rather than a fampridine-induced response or that a higher level of inflammation induces a better response to fampridine.

Comparative analysis of hippocampal extracellular space uncovers widely altered peptidome upon epileptic seizure in urethane-anaesthetized rats.

BACKGROUND: The brain extracellular fluid (ECF), composed of secreted neurotransmitters, metabolites, peptides, and proteins, may reflect brain processes. Analysis of brain ECF may provide new potential markers for synaptic activity or brain damage and reveal additional information on pathological alterations. Epileptic seizure induction is an acute and harsh intervention in brain functions, and it can activate extra- and intracellular proteases, which implies an altered brain secretome. Thus, we applied a 4-aminopyridine (4-AP) epilepsy model to study the hippocampal ECF peptidome alterations upon treatment in rats. METHODS: We performed in vivo microdialysis in the hippocampus for 3-3 h of control and 4-AP treatment phase in parallel with electrophysiology measurement. Then, we analyzed the microdialysate peptidome of control and treated samples from the same subject by liquid chromatography-coupled tandem mass spectrometry. We analyzed electrophysiological and peptidomic alterations upon epileptic seizure induction by two-tailed, paired t-test. RESULTS: We detected 2540 peptides in microdialysate samples by mass spectrometry analysis; and 866 peptides-derived from 229 proteins-were found in more than half of the samples. In addition, the abundance of 322 peptides significantly altered upon epileptic seizure induction. Several proteins of significantly altered peptides are neuropeptides (Chgb) or have synapse- or brain-related functions such as the regulation of synaptic vesicle cycle (Atp6v1a, Napa), astrocyte morphology (Vim), and glutamate homeostasis (Slc3a2). CONCLUSIONS: We have detected several consequences of epileptic seizures at the peptidomic level, as altered peptide abundances of proteins that regulate epilepsy-related cellular processes. Thus, our results indicate that analyzing brain ECF by in vivo microdialysis and omics techniques is useful for monitoring brain processes, and it can be an alternative method in the discovery and analysis of CNS disease markers besides peripheral fluid analysis.

Measuring and predicting the effect of remyelinating therapy in multiple sclerosis: a randomised controlled trial protocol (RESTORE).

INTRODUCTION: Remyelination failure hampers symptomatic recovery in multiple sclerosis (MS), underlining the importance of developing remyelinating therapies. Optic neuritis is currently the most established method of measuring remyelination in MS trials. Complementary more generalisable methods of measuring remyelination are required to confirm treatment efficacy. Measuring internuclear ophthalmoplegia (INO) with infrared oculography provides such a method. Moreover, this method can be expanded with a test for selecting likely treatment responders by using fampridine. The aim of this trial is to investigate the (long-term) remyelinating effects of clemastine fumarate in patients with MS and INO and to evaluate if treatment response can be predicted using fampridine. METHODS AND ANALYSIS: RESTORE is a single-centre double-blind randomised placebo-controlled trial of clemastine fumarate versus placebo. Prior to clemastine treatment improvement in oculographic features of INO after a single 10 mg dose of fampridine is measured in all participants and used to predict the treatment response to clemastine. Eighty individuals with MS and INO will be 1:1 randomised to 4 mg of clemastine fumarate two times a day for 6 months or equivalent placebo. Our primary outcome is improvement in the Versional Dysconjugacy Index-area under the curve, measured by infrared oculography after 6 months of treatment. Participants are assessed for persistent treatment effects 6, 18 and 30 months after end of treatment. Secondary outcome measures include other oculography parameters including double-step saccades, retinal imaging, visual acuities, physical disability, cognition and patient-reported outcomes. ETHICS AND DISSEMINATION: Clemastine is a registered and very well-established drug with well-known safety and side effects. The protocol was approved by the medical ethical committee of the Amsterdam UMC, location VUMC and the Dutch Central Committee on Research Involving Human Subject. Written informed consent is obtained from all participants. The results will be published in peer-reviewed medical scientific journals. TRIAL REGISTRATION NUMBER: EudraCT: 2021-003677-66, ClinicalTrials.gov: NCT05338450.

Cisplatin and Procaterol Combination in Gastric Cancer? Targeting Checkpoint Kinase 1 for Cancer Drug Discovery and Repurposing by an Integrated Computational and Experimental Approach.

Checkpoint kinase 1 (CHK1), a serine/threonine kinase, plays a crucial role in cell cycle arrest and is a promising therapeutic target for drug development against cancers. CHK1 coordinates cell cycle checkpoints in response to DNA damage, facilitating repair of single-strand breaks, and maintains the genome integrity in response to replication stress. In this study, we employed an integrated computational and experimental approach to drug discovery and repurposing, aiming to identify a potent CHK1 inhibitor among existing drugs. An e-pharmacophore model was developed based on the three-dimensional crystal structure of the CHK1 protein in complex with CCT245737. This model, characterized by seven key molecular features, guided the screening of a library of drugs through molecular docking. The top 10% of scored ligands were further examined, with procaterol emerging as the leading candidate. Procaterol demonstrated interaction patterns with the CHK1 active site similar to CHK1 inhibitor (CCT245737), as shown by molecular dynamics analysis. Subsequent in vitro assays, including cell proliferation, colony formation, and cell cycle analysis, were conducted on gastric adenocarcinoma cells treated with procaterol, both as a monotherapy and in combination with cisplatin. Procaterol, in synergy with cisplatin, significantly inhibited cell growth, suggesting a potentiated therapeutic effect. Thus, we propose the combined application of cisplatin and procaterol as a novel potential therapeutic strategy against human gastric cancer. The findings also highlight the relevance of CHK1 kinase as a drug target for enhancing the sensitivity of cytotoxic agents in cancer.

Ursolic acid inhibits the synaptic release of glutamate and prevents glutamate excitotoxicity in rats.

The present study evaluated the effect of ursolic acid, a natural pentacyclic triterpenoid, on glutamate release in rat cortical nerve terminals (synaptosomes) and its neuroprotection in a kainic acid-induced excitotoxicity rat model. In cortical synaptosomes, ursolic acid produced a concentration-dependent inhibition of evoked glutamate release with a half-maximum inhibition of release value of 9.5 µM, and calcium-free medium and the P/Q -type Ca2+ channel blocker, ω-agatoxin IVA, but not ω-conotoxin GVIA, an N-type Ca2+ channel blocker, prevented the ursoloic acid effect. The molecular docking study indicated that ursolic acid interacted with P/Q-type Ca2+ channels. Ursolic acid also significantly decreased the depolarization-induced activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and the subsequent phosphorylation of synapsin I, and the ursolic acid effect on evoked glutamate release was inhibited by the CaMKII inhibitor KN 62 in synaptosomes. In addition, in rats that were intraperitoneally injected with ursolic acid 30 min before kainic acid intraperitoneal injection, cortical neuronal degeneration was attenuated. This effect of ursolic acid in the improvement of kainic acid-induced neuronal damage was associated with the reduction of kainic acid-induced glutamate increase in the cortex of rats; this was characterized by the reduction of glutamate and glutaminase levels and elevation of glutamate dehydrogenase, glutamate transporter 1, glutamate-aspartate transporter, and glutamine synthetase protein levels. These results suggest that ursolic acid inhibits glutamate release from cortical synaptosomes by decreasing P/Q-type Ca2+ channel activity and subsequently suppressing CaMKII and exerts a preventive effect against glutamate neurotoxicity by controlling glutamate levels.

Cell-specific NFIA upregulation promotes epileptogenesis by TRPV4-mediated astrocyte reactivity.

BACKGROUND: The astrocytes in the central nervous system (CNS) exhibit morphological and functional diversity in brain region-specific pattern. Functional alterations of reactive astrocytes are commonly present in human temporal lobe epilepsy (TLE) cases, meanwhile the neuroinflammation mediated by reactive astrocytes may advance the development of hippocampal epilepsy in animal models. Nuclear factor I-A (NFIA) may regulate astrocyte diversity in the adult brain. However, whether NFIA endows the astrocytes with regional specificity to be involved in epileptogenesis remains elusive. METHODS: Here, we utilize an interference RNA targeting NFIA to explore the characteristics of NFIA expression and its role in astrocyte reactivity in a 4-aminopyridine (4-AP)-induced seizure model in vivo and in vitro. Combined with the employment of a HA-tagged plasmid overexpressing NFIA, we further investigate the precise mechanisms how NIFA facilitates epileptogenesis. RESULTS: 4-AP-induced NFIA upregulation in hippocampal region is astrocyte-specific, and primarily promotes detrimental actions of reactive astrocyte. In line with this phenomenon, both NFIA and vanilloid transient receptor potential 4 (TRPV4) are upregulated in hippocampal astrocytes in human samples from the TLE surgical patients and mouse samples with intraperitoneal 4-AP. NFIA directly regulates mouse astrocytic TRPV4 expression while the quantity and the functional activity of TRPV4 are required for 4-AP-induced astrocyte reactivity and release of proinflammatory cytokines in the charge of NFIA upregulation. NFIA deficiency efficiently inhibits 4-AP-induced TRPV4 upregulation, weakens astrocytic calcium activity and specific astrocyte reactivity, thereby mitigating aberrant neuronal discharges and neuronal damage, and suppressing epileptic seizure. CONCLUSIONS: Our results uncover the critical role of NFIA in astrocyte reactivity and illustrate how epileptogenic brain injury initiates cell-specific signaling pathway to dictate the astrocyte responses.

Dalfampridine as a promising agent in the management of hereditary spastic paraplegia: A triple-blinded, randomized, placebo-controlled pilot trial.

Limited but encouraging results support the use of dalfampridine in patients with hereditary spastic paraplegia (HSP). Our aim was to investigate the effects of dalfampridine on walking speed, muscle length, spasticity, functional strength, and functional mobility in patients with HSP. In this triple-blinded, randomized, placebo-controlled pilot trial, four patients with HSP received dalfampridine (10 mg twice daily) in addition to physiotherapy (twice a week), and four patients received placebo in addition to physiotherapy for eight weeks. The group allocation was masked from the assessor, treating physiotherapists, and patients. The primary outcome was the Timed 25-foot Walk Test (T25FWT) at the end of the eight-week treatment. The secondary outcome measures were functional mobility, functional muscle strength, muscle length, and spasticity. The improvement in the T25FWT values was significantly higher in the experimental group than in the control group (p < 0.05). All patients in the experimental group exceeded the proposed minimally important clinical difference for T25FWT. The degrees of improvement in most muscle length and spasticity assessments and functional muscle strength were also higher in the experimental group (p < 0.05). No significant difference was observed between the groups regarding functional mobility (p > 0.05). No adverse events or side effects were noted. This pilot trial yields encouraging evidence that the combination of dalfampridine and physiotherapy may enhance muscle parameters and improve walking speed in patients with HSP. However, further research involving larger sample sizes and more comprehensive assessments is needed to validate these results and establish the clinical benefits of this treatment approach. Trial registration ID: NCT05613114 (https://clinicaltrials.gov/), retrospectively registered on November 14, 2022.

Synthesis and Biological Study of 4-Aminopyridine-Peptide Derivatives Designed for the Treatment of Neurodegenerative Disorders.

BACKGROUND: Alzheimer's disease (AD) and Multiple sclerosis (MS) lead to neurodegenerative processes negatively affecting millions of people worldwide. Their treatment is still difficult and practically incomplete. One of the most commonly used drugs against these neurodegenerative diseases is 4-aminopyridine. However, its use is confined by the high toxicity. OBJECTIVES: The aim of this work is to obtain new peptide derivatives of 4-aminopyridine with decreased toxicity compared to 4-aminopyridine. METHODS: Synthesis was conducted in solution using a consecutive condensation approach. The new derivatives were characterized by melting points, NMR, and Mass spectra. Important ADME (absorption, distribution, metabolism, and excretion) properties have been studied in silico using ACD/Percepta v.2020.2.0 software. Acute toxicity was determined in mice according to a Standard protocol. All new derivatives were tested in vitro for cytotoxic activity in a panel of human (HEP-G2, BV-173) and murine (NEURO 2A) tumor cell lines via a standard MTT-based colorimetric method. ß-secretase inhibitory activity was determined by applying the fluorescent method. RESULTS: New derivatives of 4-aminopyridine containing analogues of the ß-secretase inhibitory peptide (Boc-Val-Asn-Leu-Ala-OH) were obtained. The in vivo toxicity of the tested compounds was found to be as high as 1500 mg/kg. Cell toxicity screening against tumor cell lines of different origins showed negligible growth-inhibitory effects of all investigated 4-aminopyridine analogues. CONCLUSION: Synthesis of new peptide derivatives of 4-aminopyridine is reported. Acute toxicity studies revealed a ca. 150 times lower toxicity of the new compounds as compared to 4-aminopyridine that may be ascribed to their peptide fragment.

In vitro fertilization with frozen embryo transfer increased histamine-mediated contractile sensitivity via PKCß in human umbilical vein.

OBJECTIVE: In vitro fertilization-embryo transfer (IVF-ET) technologies (especially frozen ET) have been widely used, which might affect maternal and fetal health. Information regarding influence of IVF-ET on the vasoconstriction of human umbilical vein (HUV) is limited. This study determined effects of frozen ET on histamine-mediated vascular responses in HUV and related mechanisms. METHODS AND RESULTS: HUVs were collected from frozen ET conceived pregnancy and spontaneously conceived pregnancy (control). Histamine concentration in umbilical plasma was higher in frozen ET group than the control. Histamine-mediated contractile response curve was left-shifted in the frozen ET group when comparing with the control. In isolated HUV rings, H1R showed a critical role in regulating vascular constriction, while H2R played little roles in regulating vessel tone. Iberiotoxin and 4-aminopyridine didn't significantly change histamine-mediated constriction in HUVs. Histamine-induced vasoconstrictions were significantly decreased by nifedipine, KN93, or GF109203X, while the inhibitory effects were significantly greater in the frozen ET group in comparison to the control. The constrictions by Bay K8644, phenylephrine, or PDBu were stronger in frozen ET, respectively. There was a decrease in the protein expressions of H1R and H2R, an increase in protein expressions of BKCaα and PKCß. CONCLUSIONS: Histamine-induced constriction in HUV was mainly via H1R. The increased sensitivity to histamine in HUV following frozen ET cycles were linked to the enhanced PKCß protein expression and function. The new data and findings in this study provide important insight into influences of frozen ET on fetal vessel development and potential influence in long-term.

Mangiferin depresses vesicular glutamate release in synaptosomes from the rat cerebral cortex by decreasing synapsin I phosphorylation.

Mangiferin is a glucosyl xanthone that has been shown to be a neuroprotective agent against brain disorders involving excess glutamate. However, the effect of mangiferin on the function of the glutamatergic system has not been investigated. In this study, we used synaptosomes from the rat cerebral cortex to investigate the effect of mangiferin on glutamate release and identify the possible underlying mechanism. We observed that mangiferin produced a concentration-dependent reduction in the release of glutamate elicited by 4-aminopyridine with an IC50 value of 25 µM. Inhibition of glutamate release was blocked by removing extracellular calcium and by treatment with the vacuolar-type H+-ATPase inhibitor bafilomycin A1, which prevents the uptake and storage of glutamate in vesicles. Moreover, we showed that mangiferin decreased the 4-aminopyridine-elicited FM1-43 release and synaptotagmin 1 luminal domain antibody (syt1-L ab) uptake from synaptosomes, which correlated with decreased synaptic vesicle exocytosis. Transmission electron microscopy in synaptosomes also showed that mangiferin attenuated the 4-aminopyridine-elicited decrease in the number of synaptic vesicles. In addition, antagonism of Ca2+/calmodulin-dependent kinase II (CaMKII) and protein kinase A (PKA) counteracted mangiferin's effect on glutamate release. Mangiferin also decreased the phosphorylation of CaMKII, PKA, and synapsin I elicited by 4-aminopyridine treatment. Our data suggest that mangiferin reduces PKA and CaMKII activation and synapsin I phosphorylation, which could decrease synaptic vesicle availability and lead to a subsequent reduction in vesicular glutamate release from synaptosomes.

Cynarin, a caffeoylquinic acid derivative in artichoke, inhibits exocytotic glutamate release from rat cortical nerve terminals (synaptosomes).

The purpose of this study was to evaluate the effect of cynarin, a caffeoylquinic acid derivative in artichoke, on glutamate release elicited by 4-aminopyridine (4-AP) in rat cortical nerve terminals (synaptosomes). We observed that cynarin decreased 4-aminopyridine-elicited glutamate release, which was prevented by the removal of external free Ca2+ with ethylene glycol bis (ß-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA) or the blockade of P/Q-type calcium channels with ω-agatoxin IVA. Molecular docking also revealed that cynarin formed a hydrogen bond with the P/Q-type Ca2+ channel, indicating a mechanism of action involving Ca2+ influx inhibition. Additionally, the inhibitory effect of cynarin on glutamate release is associated with a change in the available synaptic vesicles, as cynarin decreased 4-AP-elicited FM1-43 release or hypertonic sucrose-evoked glutamate release from synaptosomes. Furthermore, the suppression of protein kinase A (PKA) prevented the effect of cynarin on 4-AP-elicited glutamate release. 4-AP-elicited PKA and synapsin I or synaptosomal-associated protein of 25 kDa (SNAP-25) phosphorylation at PKA-specific residues were also attenuated by cynarin. Our data indicate that cynarin, through the suppression of P/Q-type Ca2+ channels, inhibits PKA activation and attenuates synapsin I and SNAP-25 phosphorylation at PKA-specific residues, thus decreasing synaptic vesicle availability and contributing to glutamate release inhibition in cerebral cortex terminals.