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2.
Int J Mol Sci ; 22(19)2021 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-34638858

RESUMO

TMEM175 (transmembrane protein 175) coding sequence variants are associated with increased risk of Parkinson's disease. TMEM175 is the ubiquitous lysosomal K+ channel regulated by growth factor receptor signaling and direct interaction with protein kinase B (PKB/Akt). In the present study, we show that the expression of mouse TMEM175 results in very small K+ currents through the plasma membrane in Xenopus laevis oocytes, in good accordance with the previously reported intracellular localization of the channel. However, the application of the dynamin inhibitor compounds, dynasore or dyngo-4a, substantially increased TMEM175 currents measured by the two-electrode voltage clamp method. TMEM175 was more permeable to cesium than potassium ions, voltage-dependently blocked by 4-aminopyridine (4-AP), and slightly inhibited by extracellular acidification. Immunocytochemistry experiments indicated that dyngo-4a increased the amount of epitope-tagged TMEM175 channel on the cell surface. The coexpression of dominant-negative dynamin, and the inhibition of clathrin- or caveolin-dependent endocytosis increased TMEM175 current much less than dynasore. Therefore, dynamin-independent pharmacological effects of dynasore may also contribute to the action on the channel. TMEM175 current rapidly decays after the withdrawal of dynasore, raising the possibility that an efficient internalization mechanism removes the channel from the plasma membrane. Dyngo-4a induced about 20-fold larger TMEM175 currents than the PKB activator SC79, or the coexpression of a constitutively active mutant PKB with the channel. In contrast, the allosteric PKB inhibitor MK2206 diminished the TMEM175 current in the presence of dyngo-4a. These data suggest that, in addition to the lysosomes, PKB-dependent regulation also influences TMEM175 current in the plasma membrane.


Assuntos
Membrana Celular/metabolismo , Hidrazonas/farmacologia , Lisossomos/metabolismo , Naftóis/farmacologia , Canais de Potássio/metabolismo , 4-Aminopiridina/farmacologia , Animais , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Células HEK293 , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Microscopia Confocal/métodos , Oócitos/citologia , Oócitos/metabolismo , Oócitos/fisiologia , Técnicas de Patch-Clamp/métodos , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/genética , Transporte Proteico/efeitos dos fármacos , Xenopus laevis
4.
BMC Neurol ; 21(1): 371, 2021 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-34563155

RESUMO

BACKGROUND: Lambert-Eaton myasthenic syndrome (LEMS) is a rare autoimmune disorder of neuromuscular transmission. The objective was to examine the efficacy and safety of 3,4-diaminopyridine (3,4-DAP) in patients with LEMS. METHODS: We searched several databases to identify relevant studies, including PubMed, EMBASE, Web of Science, MEDLINE, Cochrane Neuromuscular Disease Group Specialized Register and the Cochrane Central Register of Controlled Trials(CENTRAL). The primary outcome, quantitative myasthenia gravis (QMG) score and the secondary outcome, compound muscle action potentials (CMAP) amplitude were pooled by meta-analysis. RESULTS: Six randomised controlled trials (RCTs) involving 115 patients with LEMS were included. QMG score showed a significant decrease (improvement) of 2.76 points (95 % CI, -4.08 to -1.45, p < 0.001) after treatment with 3, 4-DAP. Moreover, the overall mean CMAP amplitude improved significantly in LEMS patients with 3, 4-DAP treatment, compared with placebo treatment (mean difference 1.34 mV, 95 % CI, 0.98 to 1.70, p < 0.001). The overall assessment of all included trials showed a low risk of bias and low heterogeneity. CONCLUSIONS: The pooled results of RCTs demonsrated with moderate to high evidence that 3,4-DAP has a significant effect on LEMS treatment, with improvements in muscle strength score and CMAP amplitude.


Assuntos
Síndrome Miastênica de Lambert-Eaton , Miastenia Gravis , 4-Aminopiridina/uso terapêutico , Adulto , Amifampridina , Humanos , Síndrome Miastênica de Lambert-Eaton/tratamento farmacológico , Miastenia Gravis/tratamento farmacológico , Ensaios Clínicos Controlados Aleatórios como Assunto
5.
Int J Mol Sci ; 22(18)2021 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-34576077

RESUMO

Kv1.2 channels, encoded by the KCNA2 gene, are localized in the central and peripheral nervous system, where they regulate neuronal excitability. Recently, heterozygous mutations in KCNA2 have been associated with a spectrum of symptoms extending from epileptic encephalopathy, intellectual disability, and cerebellar ataxia. Patients are treated with a combination of antiepileptic drugs and 4-aminopyridine (4-AP) has been recently trialed in specific cases. We identified a novel variant in KCNA2, E236K, in a Serbian proband with non-progressive congenital ataxia and early onset epilepsy, treated with sodium valproate. To ascertain the pathogenicity of E236K mutation and to verify its sensitivity to 4-AP, we transfected HEK 293 cells with Kv1.2 WT or E236K cDNAs and recorded potassium currents through the whole-cell patch-clamp. In silico analysis supported the electrophysiological data. E236K channels showed voltage-dependent activation shifted towards negative potentials and slower kinetics of deactivation and activation compared with Kv1.2 WT. Heteromeric Kv1.2 WT+E236K channels, resembling the condition of the heterozygous patient, confirmed a mixed gain- and loss-of-function (GoF/LoF) biophysical phenotype. 4-AP inhibited both Kv1.2 and E236K channels with similar potency. Homology modeling studies of mutant channels suggested a reduced interaction between the residue K236 in the S2 segment and the gating charges at S4. Overall, the biophysical phenotype of E236K channels correlates with the mild end of the clinical spectrum reported in patients with GoF/LoF defects. The response to 4-AP corroborates existing evidence that KCNA2-disorders could benefit from variant-tailored therapeutic approaches, based on functional studies.


Assuntos
4-Aminopiridina/uso terapêutico , Ataxia Cerebelar/congênito , Ataxia Cerebelar/genética , Epilepsia/tratamento farmacológico , Epilepsia/genética , Canal de Potássio Kv1.2/genética , Sequência de Aminoácidos , Encéfalo/diagnóstico por imagem , Ataxia Cerebelar/diagnóstico por imagem , Ataxia Cerebelar/tratamento farmacológico , Criança , Pré-Escolar , Epilepsia/diagnóstico por imagem , Humanos , Lactente , Canal de Potássio Kv1.2/química , Imageamento por Ressonância Magnética , Masculino , Simulação de Dinâmica Molecular , Adulto Jovem
6.
Biomolecules ; 11(7)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34356653

RESUMO

Rosmarinic acid, a major component of rosemary, is a polyphenolic compound with potential neuroprotective effects. Asreducing the synaptic release of glutamate is crucial to achieving neuroprotectant's pharmacotherapeutic effects, the effect of rosmarinic acid on glutamate release was investigated in rat cerebrocortical nerve terminals (synaptosomes). Rosmarinic acid depressed the 4-aminopyridine (4-AP)-induced glutamate release in a concentration-dependent manner. The removal of extracellular calcium and the blockade of vesicular transporters prevented the inhibition of glutamate release by rosmarinic acid. Rosmarinic acid reduced 4-AP-induced intrasynaptosomal Ca2+ elevation. The inhibition of N-, P/Q-type Ca2+ channels and the calcium/calmodulin-dependent kinase II (CaMKII) prevented rosmarinic acid from having effects on glutamate release. Rosmarinic acid also reduced the 4-AP-induced activation of CaMKII and the subsequent phosphorylation of synapsin I, the main presynaptic target of CaMKII. In addition, immunocytochemistry confirmed the presence of GABAA receptors. GABAA receptor agonist and antagonist blocked the inhibitory effect of rosmarinic acid on 4-AP-evoked glutamate release. Docking data also revealed that rosmarinic acid formed a hydrogen bond with the amino acid residues of GABAA receptor. These results suggested that rosmarinic acid activates GABAA receptors in cerebrocortical synaptosomes to decrease Ca2+ influx and CaMKII/synapsin I pathway to inhibit the evoked glutamate release.


Assuntos
Cinamatos/farmacologia , Depsídeos/farmacologia , Ácido Glutâmico/metabolismo , Sinaptossomos/efeitos dos fármacos , 4-Aminopiridina/farmacologia , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Cinamatos/química , Depsídeos/química , Agonistas de Receptores de GABA-A/farmacologia , Antagonistas de Receptores de GABA-A/farmacologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Simulação de Acoplamento Molecular , Fármacos Neuroprotetores/farmacologia , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Ratos Sprague-Dawley , Receptores de GABA-A/química , Receptores de GABA-A/metabolismo , Sinaptossomos/metabolismo
8.
Am J Physiol Cell Physiol ; 321(4): C684-C693, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34432539

RESUMO

Transient outward, or "A-type," currents are rapidly inactivating voltage-gated potassium currents that operate at negative membrane potentials. A-type currents have not been reported in the gastric fundus, a tonic smooth muscle. We used whole cell voltage clamp to identify and characterize A-type currents in smooth muscle cells (SMCs) isolated from murine fundus. A-type currents were robust in these cells with peak amplitudes averaging 1.5 nA at 0 mV. Inactivation was rapid with a time constant of 71 ms at 0 mV; recovery from inactivation at -80 mV was similarly rapid with a time constant of 75 ms. A-type currents in fundus were blocked by 4-aminopyridine (4-AP), flecainide, and phrixotoxin-1 (PaTX1). Remaining currents after 4-AP and PaTX1 displayed half-activation potentials that were shifted to more positive potentials and showed incomplete inactivation. Currents after tetraethylammonium (TEA) displayed half inactivation at -48.1 ± 1.0 mV. Conventional microelectrode and contractile experiments on intact fundus muscles showed that 4-AP depolarized membrane potential and increased tone under conditions in which enteric neurotransmission was blocked. These data suggest that A-type K+ channels in fundus SMCs are likely active at physiological membrane potentials, and sustained activation of A-type channels contributes to the negative membrane potentials of this tonic smooth muscle. Quantitative analysis of Kv4 expression showed that Kcnd3 was dominantly expressed in fundus SMCs. These data were confirmed by immunohistochemistry, which revealed Kv4.3-like immunoreactivity within the tunica muscularis. These observations indicate that Kv4 channels likely form the A-type current in murine fundus SMCs.


Assuntos
Fundo Gástrico/metabolismo , Motilidade Gastrointestinal , Contração Muscular , Músculo Liso/metabolismo , Potássio/metabolismo , Canais de Potássio Shal/metabolismo , 4-Aminopiridina/farmacologia , Animais , Fundo Gástrico/efeitos dos fármacos , Motilidade Gastrointestinal/efeitos dos fármacos , Cinética , Masculino , Potenciais da Membrana , Camundongos Endogâmicos BALB C , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Shal/antagonistas & inibidores , Canais de Potássio Shal/genética , Venenos de Aranha/metabolismo
9.
Exp Brain Res ; 239(9): 2841-2849, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34283252

RESUMO

Phoenixin-14 (PNX-14) has a wide bioactivity in the central nervous system. Its role in the hypothalamus has been investigated, and it has been reported that it is involved in the regulation of excitability in hypothalamic neurons. However, its role in the regulation of excitability in entorhinal cortex and the hippocampus is unknown. In this study, we investigated whether i. PNX-14 induces any synchronous discharges or epileptiform activity and ii. PNX-14 has any effect on already initiated epileptiform discharges. We used 350 µm thick acute horizontal hippocampal-entorhinal cortex slices obtained from 30- to 35-day-old mice. Extracellular field potential recordings were evaluated in the entorhinal cortex and hippocampus CA1 region. Bath application of PNX-14 did not initiate any epileptiform activity or abnormal discharges. 4-Aminopyridine was applied to induce epileptiform activity in the slices. We found that 200 nM PNX-14 reduced the frequency of interictal-like events in both the entorhinal cortex and hippocampus CA1 region which was induced by 4-aminopyridine. Furthermore, PNX-14 led to a similar suppression in the total power of local field potentials of 1-120 Hz. The frequency or the duration of the ictal events was not affected. These results exhibited for the first time that PNX-14 has a modulatory effect on synchronized neuronal discharges which should be considered in future therapeutic approaches.


Assuntos
Córtex Entorrinal , Hipocampo , 4-Aminopiridina/farmacologia , Animais , Camundongos , Neurônios
10.
World Neurosurg ; 153: e168-e178, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34166824

RESUMO

PURPOSE: To study the 24-hour expression of long noncoding RNAs (lncRNAs) in synaptic and extrasynaptic neurons expressing N-methyl-D-aspartate receptor (NMDAR), and normal neuronal cultures, via microarray analysis. MATERIALS AND METHODS: Cortical neurons from embryonic (day E18) Sprague-Dawley rats were used for primary neuronal culture. NMDAR activation was blocked and the cells were then incubated for 6 hours. Total RNA was extracted, quantified, and analyzed for purity and integrity. Double-stranded cDNA was synthesized, followed by quantile normalization, quantitative polymerase chain reaction validation, and data analysis. The interactions between transcription factors and lncRNAs were analyzed by Pearson correlation. RESULTS: The lncRNA profiles were obtained after synaptic and extrasynaptic NMDAR activation of rat cortical neuron cultures for 24 hours. In total, 251 lncRNAs were consistently upregulated, and 335 were downregulated, after extrasynaptic NMDAR activation compared with normal neurons. After synaptic NMDAR activation, only 9 lncRNAs were upregulated and 2 were downregulated. CONCLUSIONS: Differential expression of lncRNAs after synaptic and extrasynaptic NMDAR activation suggests that lncRNAs may be responsible for extrasynaptic NMDAR-induced neurodegeneration.


Assuntos
Neurônios/metabolismo , RNA Longo não Codificante/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/metabolismo , 4-Aminopiridina/farmacologia , Animais , Bicuculina/farmacologia , Córtex Cerebral/citologia , Regulação para Baixo , Agonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Receptores de GABA-A/farmacologia , Glicina/farmacologia , Glicinérgicos/farmacologia , Análise em Microsséries , N-Metilaspartato/farmacologia , Reação em Cadeia da Polimerase , Bloqueadores dos Canais de Potássio/farmacologia , Cultura Primária de Células , Ratos , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Regulação para Cima
11.
Epilepsia ; 62(7): 1505-1517, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33979453

RESUMO

OBJECTIVE: One of the challenges in treating patients with drug-resistant epilepsy is that the mechanisms of seizures are unknown. Most current interventions are based on the assumption that epileptic activity recruits neurons and progresses by synaptic transmission. However, several experimental studies have shown that neural activity in rodent hippocampi can propagate independently of synaptic transmission. Recent studies suggest these waves are self-propagating by electric field (ephaptic) coupling. In this study, we tested the hypothesis that neural recruitment during seizures can occur by electric field coupling. METHODS: 4-Aminopyridine was used in both in vivo and in vitro preparation to trigger seizures or epileptiform activity. A transection was made in the in vivo hippocampus and in vitro hippocampal and cortical slices to study whether the induced seizure activity can recruit neurons across the gap. A computational model was built to test whether ephaptic coupling alone can account for neural recruitment across the transection. The model prediction was further validated by in vitro experiments. RESULTS: Experimental results show that electric fields generated by seizure-like activity in the hippocampus both in vitro and in vivo can recruit neurons locally and through a transection of the tissue. The computational model suggests that the neural recruitment across the transection is mediated by electric field coupling. With in vitro experiments, we show that a dielectric material can block the recruitment of epileptiform activity across a transection, and that the electric fields measured within the gap are similar to those predicted by model simulations. Furthermore, this nonsynaptic neural recruitment is also observed in cortical slices, suggesting that this effect is robust in brain tissue. SIGNIFICANCE: These results indicate that ephaptic coupling, a nonsynaptic mechanism, can underlie neural recruitment by a small electric field generated by seizure activity and could explain the low success rate of surgical transections in epilepsy patients.


Assuntos
Campos Eletromagnéticos , Epilepsia/fisiopatologia , Recrutamento Neurofisiológico , 4-Aminopiridina , Animais , Córtex Cerebral/fisiopatologia , Simulação por Computador , Convulsivantes , Epilepsia/diagnóstico , Feminino , Hipocampo/fisiopatologia , Masculino , Camundongos Transgênicos , Modelos Neurológicos , Valor Preditivo dos Testes , Ratos , Ratos Sprague-Dawley , Convulsões/diagnóstico , Convulsões/fisiopatologia , Transmissão Sináptica
12.
Clin Biomech (Bristol, Avon) ; 86: 105382, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34000628

RESUMO

BACKGROUND: People with multiple sclerosis have reduced walking speed and impaired gait pattern. Prolonged release-fampridine is a potassium channel blocker that improves nerve conduction in patients with multiple sclerosis, leading to walking benefits. Whether fampridine alters gait pattern is unknown. METHODS: In this crossover, randomized controlled trial, patients with multiple sclerosis were tested for responder status during a 4-week run-in period. Patients were considered responders if they improved their 25-ft walk test by 10% and improved their perceived walking capacity. Responders were randomized to prolonged release-fampridine (10 mg b.i.d.) or placebo for a 6-week period. After a 2-week wash-out period, they were allocated to the other treatment for 6 weeks. Participants were assessed before and after both conditions. Three-dimensional gait analysis assessed kinematic, kinetic, mechanic and energetic variables while walking on a treadmill at comfortable speed. Six-minute walk test and 25-ft walk test were used to assess walking speed on middle and short-distances, respectively. Patient-reported outcome measures were also used. Repeated measures ANCOVAs were applied to assess the treatment effects. FINDINGS: Out of 39 included patients, 24 responders (12 women; Expanded Disability Status Scale:4.25[4-5]; age:46 ± 10 years; maximal speed:0.93 ± 0.38 m·s-1) were identified. Among them, prolonged release-fampridine reduced the external mechanical work (-0.039 J·kg-1·m-1;p = 0.02), and improved knee flexion during swing phase (+5.3°; p = 0.02). No differences were found in other walking tests and patient-reported outcomes, at group-level. INTERPRETATION: Prolonged release-fampridine increases knee flexion during swing phase and lowers mechanical external work. Whether these changes are related to clinically meaningful improvements in walking capacity and other functional variables should be further investigated.


Assuntos
Esclerose Múltipla , 4-Aminopiridina/farmacologia , 4-Aminopiridina/uso terapêutico , Adulto , Feminino , Marcha , Humanos , Pessoa de Meia-Idade , Esclerose Múltipla/complicações , Esclerose Múltipla/tratamento farmacológico , Bloqueadores dos Canais de Potássio/farmacologia , Bloqueadores dos Canais de Potássio/uso terapêutico , Resultado do Tratamento , Caminhada
13.
J Physiol ; 599(12): 3195-3220, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33942325

RESUMO

KEY POINTS: Extracellular space (ECS) rapid volume pulsation (RVP) accompanying epileptiform activity is described for the first time. Such RVP occurs robustly in several in vitro and in vivo mouse models of epileptiform activity. In the in vitro 4-aminopyridine model of epileptiform activity, RVP depends on the activity of the electrogenic Na+ /HCO3 - cotransporter (NBCe1). NBCe1 pharmacological inhibition suppresses RVP and epileptiform activity. Inhibition of changes in ECS volume may be a useful target in epilepsy patients who are resistant to current treatments. ​ ABSTRACT: The extracellular space (ECS) of the brain shrinks persistently by approximately 35% during epileptic seizures. Here we report the discovery of rapid volume pulsation (RVP), further transient drops in ECS volume which accompany events of epileptiform activity. These transient ECS contractions were observed in multiple mouse models of epileptiform activity both in vivo (bicuculline methiodide model) and in vitro (hyaluronan synthase 3 knock-out, picrotoxin, bicuculline and 4-aminopyridine models). By using the probe transients quantification (PTQ) method we show that individual pulses of RVP shrank the ECS by almost 15% in vivo. In the 4-aminopyridine in vitro model, the individual pulses of RVP shrank the ECS by more than 4%, and these transient changes were superimposed on a persistent ECS shrinkage of 36% measured with the real-time iontophoretic method. In this in vitro model, we investigated several channels and transporters that may be required for the generation of RVP and epileptiform activity. Pharmacological blockages of Na+ /K+ /2Cl- cotransporter type 1 (NKCC1), K+ /Cl- cotransporter (KCC2), the water channel aquaporin-4 (AQP4) and inwardly rectifying potassium channel 4.1 (Kir4.1) were ineffective in halting the RVP and the epileptiform activity. In contrast, pharmacological blockade of the electrogenic Na+ /HCO3 - cotransporter (NBCe1) by 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) eliminated both the RVP and the persistent ECS shrinkage. Importantly, this blocker also stopped the epileptiform activity. These results demonstrate that RVP is closely associated with epileptiform activity across several models of epileptiform activity and therefore the underlying mechanism could potentially represent a novel target for epilepsy management and treatment.


Assuntos
Epilepsia , Espaço Extracelular , 4-Aminopiridina/farmacologia , Animais , Encéfalo/metabolismo , Epilepsia/tratamento farmacológico , Espaço Extracelular/metabolismo , Humanos , Camundongos , Simportadores de Sódio-Bicarbonato/metabolismo
14.
Neuroscience ; 466: 235-247, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-33961962

RESUMO

Convulsive status epilepticus (SE) in immature life is often associated with lasting neurobiological changes. We provoked SE by pentylenetetrazole in postnatal day 20 rat pups and examined communication modalities between the temporal hippocampus and medial entorhinal cortex (mEC) in vitro. After a minimum of 40 days post-SE, we prepared combined temporal hippocampal - medial entorhinal cortex (mEC) slices from conditioned (SE) and naïve (N) adult rats and recorded 4-aminopyridine-induced spontaneous epileptiform interictal-like discharges (IED) simultaneously from CA3 and mEC layer V-VI. We analyzed IED frequency and high frequency oscillations (HFOs) in intact slices and after surgical separation of hippocampus from mEC, by two successive incisions (Schaffer collateral cut, Parasubiculum cut). In all slices, IED frequency was higher in CA3 vs mEC (5N, 4SE) and Raster plots indicated no temporal coincidence between them either in intact or in CA1-cut slices (4N, 4SE). IED frequency was significantly higher in SE mEC, but similar in SE and N CA3, independently of connectivity state. Ripples (R) and Fast Ripples (FR) coincided with IEDs and their power differed between SE and N intact slices (22N, 12SE), both in CA3 and mEC. CA3 FR/R ratios were higher in the absence of mEC (14N, 8SE). Moreover, SE (vs N) slices showed significantly higher FR/R ratios independently of the presence of mEC. Taken together, these findings suggest lasting effects of immature SE in network dynamics governing hippocampal-entorhinal communication which may impact adult cognitive, behavioral, and/or seizure threshold sequalae.


Assuntos
Córtex Entorrinal , Estado Epiléptico , 4-Aminopiridina , Animais , Hipocampo , Pentilenotetrazol , Ratos , Estado Epiléptico/induzido quimicamente
15.
Exp Neurol ; 343: 113776, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34058228

RESUMO

Malformations of cortical development (MCD) represent a group of rare diseases with severe clinical presentation as epileptic and pharmacoresistant encephalopathies. Morphological studies in tissue from MCD patients have revealed reduced GABAergic efficacy and increased intracellular chloride concentration in neuronal cells as important pathophysiological mechanisms in MCD. Also, in various animal models, alterations of GABAergic inhibition have been postulated as a predominant factor contributing to perilesional hyperexcitability. Along with this line, the NKCC1 inhibitor bumetanide has been postulated as a potential drug for treatment of epilepsy, mediating its antiepileptic effect by reduction of the intracellular chloride and increased inhibitory efficacy of GABAergic transmission. In the present study, we focused on the focal freeze-lesion model of MCD to compare antiepileptic drugs with distinct mechanisms of action, including NKCC1 inhibition by bumetanide. For this purpose, we combined electrophysiological and optical methods in slice preparations and assessed the properties of seizure like events (SLE) induced by 4-aminopyridine. In freeze-lesioned but not control slices, SLE onset was confined to the perilesional area, confirming that this region is hyperexcitable and likely triggers pathological activity. Bumetanide selectively reduced epileptic activity in lesion-containing slices but not in slices from sham-treated control rats. Moreover, bumetanide caused a shift in the SLE onset site away from the perilesional area. In contrast, effects of other antiepileptic drugs including carbamazepine, lacosamide, acezatolamide and zonisamide occurred mostly independently of the lesion and did not result in a shift of the onset region. Our work adds evidence for the functional relevance of chloride homeostasis in the pathophysiology of microgyrus formation as represented in the focal freeze-lesion model. Further studies in different MCD models and human tissue will be required to validate the effects across different MCD subtypes and species and to assess the translational value of our findings.


Assuntos
Anticonvulsivantes/administração & dosagem , Criocirurgia/efeitos adversos , Malformações do Desenvolvimento Cortical/tratamento farmacológico , Malformações do Desenvolvimento Cortical/patologia , Convulsões/tratamento farmacológico , Convulsões/patologia , 4-Aminopiridina/toxicidade , Animais , Bumetanida/administração & dosagem , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/patologia , Masculino , Malformações do Desenvolvimento Cortical/etiologia , Técnicas de Cultura de Órgãos , Ratos , Ratos Wistar , Convulsões/induzido quimicamente , Inibidores de Simportadores de Cloreto de Sódio e Potássio/administração & dosagem
16.
Acc Chem Res ; 54(9): 2313-2322, 2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33847478

RESUMO

Life emerges from complicated and sophisticated chemical networks comprising numerous biomolecules (e.g., nucleic acids, proteins, sugars, and lipids) and chemical reactions catalyzed by enzymes. Dysregulation of these chemical networks is linked to the emergence of diseases. Our research goal is to develop abiotic chemical catalysts that can intervene into life's chemical networks by complementing, surrogating, or exceeding enzymes in living cells or multicellular organisms such as animals or plants. Mending dysregulated networks in pathological states by the chemical catalysts will lead to a new medicinal strategy, catalysis medicine. This research direction will also advance catalysis science, because highly active and selective chemical catalysts must be developed to promote the intended reactions in a complex mixture of life in aqueous solution at body temperature.Epigenetics exists at the crossroads of chemistry, biology, and medicine and is a suitable field to pursue this idea. Post-translational modifications (PTMs) of histones epigenetically regulate chromatin functions and gene transcription and are intimately related to various diseases. Investigating the functions and cross-talk of histone PTMs is crucial for mechanistic elucidation of diseases and their treatments. We launched a program to develop chemical catalysts enabling endogenous histone modifications in living cells without relying on enzymes. We reported two types of chemical catalyst systems so far for synthetic histone acylation. The first system comprised a DNA-binding oligo-4-dimethylaminopyridine (DMAP) catalyst and a phenyl ester acyl donor, PAc-gly. This system promoted histone hyperacetylation in Xenopus laevis sperm chromatin. Using the thus-synthesized hyperacetylated sperm chromatin, we found a novel relationship between histone acetylation and DNA replication. The second system involved a histone-binding catalyst, LANA-DSH, composed of a catalytic motif (DSH) and a histone-binding peptide ligand (LANA), and thioester acyl donors, including endogenous acyl-CoA. This system regioselectively (i.e., selectively to a lysine residue at a specific position) acylated lysine 120 of histone H2B (H2BK120), a lysine residue proximal to the DSH motif defined by binding of the LANA ligand to a nucleosome substrate. This catalyst system was optimized to achieve H2BK120-selective acetylation in living cells without genetic manipulation. The synthetically introduced H2BK120Ac inhibited enzyme-catalyzed ubiquitination at the same lysine residue, acting as a protecting group. H2BK120Ub is a mark recognized by methyltransferase that plays an essential role in mixed-lineage leukemia (MLL)-rearranged leukemia, suggesting the potential of the catalyst system as an epigenetic tool and a cancer therapy. We also discuss the prospects of chemical catalyst-promoted synthetic epigenetics for future PTM studies and therapeutic uses.


Assuntos
4-Aminopiridina/análogos & derivados , DNA/química , Ésteres/química , Histonas/química , Peptídeos/química , Compostos de Sulfidrila/química , 4-Aminopiridina/química , Animais , Catálise , Epigênese Genética/genética , Histonas/genética , Histonas/metabolismo , Humanos , Processamento de Proteína Pós-Traducional
17.
Eur J Neurol ; 28(7): 2249-2258, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33852752

RESUMO

BACKGROUND AND PURPOSE: Fatigue in multiple sclerosis (MS) is common and disabling; medication efficacy is still not fully proven. The aim of this study was to investigate 4-week modifications of fatigue severity in 45 relapsing-remitting MS patients after different symptomatic treatments, and changes in concomitant resting state (RS) functional connectivity (FC). METHODS: Patients were randomly, blindly assigned to treatment with fampridine (n = 15), amantadine (n = 15) or placebo (n = 15), and underwent clinical assessment and 3-Tesla RS functional magnetic resonance imaging at baseline (t0) and after 4 weeks (w4) of treatment. Fifteen healthy controls (HCs) were also studied. Changes in modified fatigue impact scale (MFIS) score and network RS FC were assessed. RESULTS: In MS, abnormalities of network RS FC at t0 did not differ between treatment groups and correlated with fatigue severity. At w4, global scores and subscores on the MFIS decreased in all groups, with no time-by-treatment interaction. At w4, all patient groups had changes in RS FC in several networks, with significant time-by-treatment interactions in basal ganglia, sensorimotor and default-mode networks in fampridine-treated patients versus the other groups, and in frontoparietal network in amantadine-treated patients. In the fampridine group, RS FC changes correlated with concurrently decreased MFIS score (r range = -0.75 to 0.74, p range = 0.003-0.05). CONCLUSIONS: Fatigue improved in all MS groups, independently of treatment. Concomitant RS FC modifications were located in sensorimotor, inferior frontal and subcortical regions for fampridine- and amantadine-treated patients, and in associative sensory cortices for placebo-treated patients.


Assuntos
Esclerose Múltipla , 4-Aminopiridina , Amantadina/uso terapêutico , Encéfalo , Fadiga/tratamento farmacológico , Fadiga/etiologia , Humanos , Imageamento por Ressonância Magnética , Esclerose Múltipla/complicações , Esclerose Múltipla/diagnóstico por imagem , Esclerose Múltipla/tratamento farmacológico
18.
Nat Commun ; 12(1): 2206, 2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33850125

RESUMO

Spreading depression (SD) is an intense and prolonged depolarization in the central nervous systems from insect to man. It is implicated in neurological disorders such as migraine and brain injury. Here, using an in vivo mouse model of focal neocortical seizures, we show that SD may be a fundamental defense against seizures. Seizures induced by topical 4-aminopyridine, penicillin or bicuculline, or systemic kainic acid, culminated in SDs at a variable rate. Greater seizure power and area of recruitment predicted SD. Once triggered, SD immediately suppressed the seizure. Optogenetic or KCl-induced SDs had similar antiseizure effect sustained for more than 30 min. Conversely, pharmacologically inhibiting SD occurrence during a focal seizure facilitated seizure generalization. Altogether, our data indicate that seizures trigger SD, which then terminates the seizure and prevents its generalization.


Assuntos
Depressão , Convulsões/induzido quimicamente , Convulsões/tratamento farmacológico , Convulsões/metabolismo , 4-Aminopiridina , Animais , Bicuculina/farmacologia , Tronco Encefálico , Depressão Alastrante da Atividade Elétrica Cortical , Feminino , Técnicas de Introdução de Genes , Ácido Caínico/farmacologia , Masculino , Camundongos , Sistema Nervoso , Optogenética , Penicilinas/farmacologia , Bloqueadores dos Canais de Potássio/efeitos adversos , Convulsões/patologia , Tetrodotoxina/farmacologia
19.
Laeknabladid ; 107(4): 179-184, 2021 Apr.
Artigo em Islandês | MEDLINE | ID: mdl-33769308

RESUMO

INTRODUCTION: Fampridine is a drug for people with Multiple Sclerosis (MS). It is a broad-spectrum voltage-dependent potassium channel blocker that enhances synaptic transmission. The drug has been shown to be able to enhance conduction in demyelinated axons, thereby leading to improved gait in patients with MS. The purpose of this study was to examine the effect of fampridine on gait function in people with MS in the end of a 2 weeks trial drug period and to observe how many patients continued drug therapy. MATERIAL AND METHODS: Data from 41 individuals with MS was collected retrospectively for this study. Measurements were administered by physiotherapists and the results from the Timed 25-Foot Walk (T25FW) and 12-item Multiple Sclerosis Walking Scale (MSWS-12) were obtained from medical records from The National University Hospital of Iceland. RESULTS: The results showed a significant difference in walking speed before and at the end of trial period (p<0.0001). The average improvement in walking speed was 22%. Results also demonstrated a significant difference in MSWS-12 scores before and at the end of treatment (p<0.0001). The average improvement in MSWS-12 was 11.4 points. Eighteen individuals (43.9%) continued treatment after the trial period. CONCLUSION: Fampridine can have a positive effect on impaired gait function in people with MS and can be an important adjunct to treatment.


Assuntos
Esclerose Múltipla , 4-Aminopiridina/efeitos adversos , Marcha , Humanos , Esclerose Múltipla/diagnóstico , Esclerose Múltipla/tratamento farmacológico , Bloqueadores dos Canais de Potássio/efeitos adversos , Estudos Retrospectivos , Caminhada
20.
Chemphyschem ; 22(10): 960-967, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-33738893

RESUMO

Signal Amplification by Reversible Exchange (SABRE) technique enables nuclear spin hyperpolarization of wide range of compounds using parahydrogen. Here we present the synthetic approach to prepare 15 N-labeled [15 N]dalfampridine (4-amino[15 N]pyridine) utilized as a drug to reduce the symptoms of multiple sclerosis. The synthesized compound was hyperpolarized using SABRE at microtesla magnetic fields (SABRE-SHEATH technique) with up to 2.0 % 15 N polarization. The 7-hour-long activation of SABRE pre-catalyst [Ir(IMes)(COD)Cl] in the presence of [15 N]dalfampridine can be remedied by the use of pyridine co-ligand for catalyst activation while retaining the 15 N polarization levels of [15 N]dalfampridine. The effects of experimental conditions such as polarization transfer magnetic field, temperature, concentration, parahydrogen flow rate and pressure on 15 N polarization levels of free and equatorial catalyst-bound [15 N]dalfampridine were investigated. Moreover, we studied 15 N polarization build-up and decay at magnetic field of less than 0.04 µT as well as 15 N polarization decay at the Earth's magnetic field and at 1.4 T.


Assuntos
4-Aminopiridina/química , 4-Aminopiridina/síntese química , Campos Magnéticos , Espectroscopia de Ressonância Magnética , Isótopos de Nitrogênio
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