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Intrahippocampal pilocarpine microinjection (H-PILO) induces status epilepticus (SE) that can lead to spontaneous recurrent seizures (SRS) and neurodegeneration in rodents. Studies using animal models have indicated that lectins mediate a variety of biological activities with neuronal benefits, especially galectin-1 (GAL-1), which has been identified as an effective neuroprotective compound. GAL-1 is associated with the regulation of cell adhesion, proliferation, programmed cell death, and immune responses, as well as attenuating neuroinflammation. Here, we administrated GAL-1 to Wistar rats and evaluated the severity of the SE, neurodegenerative and inflammatory patterns in the hippocampal formation. Administration of GAL-1 caused a reduction in the number of class 2 and 4 seizures, indicating a decrease in seizure severity. Furthermore, we observed a reduction in inflammation and neurodegeneration 24 h and 15 days after SE. Overall, these results suggest that GAL-1 has a neuroprotective effect in the early stage of epileptogenesis and provides new insights into the roles of exogenous lectins in temporal lobe epilepsy (TLE).

Evidence supports that the gut microbiota and bacteria-dependent metabolites influence the maintenance of epileptic brain activity. However, the alterations in the gut microbiota between epileptic versus healthy individuals are poorly understood. We used a multi-omic approach to evaluate the changes in the composition of gut metagenome as well in the fecal metabolomic profile in rats before and after being submitted to status epilepticus (SE)-induced temporal lobe epilepsy (TLE). The 16S ribosomal RNA (rRNA) sequencing of fecal samples coupled to bioinformatic analysis revealed taxonomic, compositional, and functional shifts in epileptic rats. The species richness (Chao1 index) was significantly lower in the post-TLE group, and the β-diversity analysis revealed clustering separated from the pre-TLE group. The taxonomic abundance analysis showed a significant increase of phylum Desulfobacterota and a decrease of Patescibacteria in the post-TLE group. The DESEq2 and LEfSe analysis resulted in 18 genera significantly enriched between post-TLE and pre-TLE groups at the genus level. We observed that epileptic rats present a peculiar metabolic phenotype, including a lower concentration of D-glucose and L-lactic acid and a higher concentration of L-glutamic acid and glycine. The microbiota-host metabolic correlation analysis showed that the genera differentially abundant in post-TLE rats are associated with the altered metabolites, especially the proinflammatory Desulfovibrio and Marvinbryantia, which were enriched in epileptic animals and positively correlated with these excitatory neurotransmitters and carbohydrate metabolites. Therefore, our data revealed a correlation between dysbacteriosis in epileptic animals and fecal metabolites that are known to be relevant for maintaining epileptic brain activity by enhancing chronic inflammation, an excitatory-inhibitory imbalance, and/or a metabolic disturbance. These data are promising and suggest that targeting the gut microbiota could provide a novel avenue for preventing and treating acquired epilepsy. However, the causal relationship between these microbial/metabolite components and the SRS occurrence still needs further exploration.

Status epilepticus (SE) can lead to serious neuronal damage and act as an initial trigger for epileptogenic processes that may lead to temporal lobe epilepsy (TLE). Besides promoting neurodegeneration, neuroinflammation, and abnormal neurogenesis, SE can generate an extensive hypometabolism in several brain areas and, consequently, reduce intracellular energy supply, such as adenosine triphosphate (ATP) molecules. Although some antiepileptic drugs show efficiency to terminate or reduce epileptic seizures, approximately 30% of TLE patients are refractory to regular antiepileptic drugs (AEDs). Modulation of glucose availability may provide a novel and robust alternative for treating seizures and neuronal damage that occurs during epileptogenesis; however, more detailed information remains unknown, especially under hypo- and hyperglycemic conditions. Here, we review several pathways of glucose metabolism activated during and after SE, as well as the effects of hypo- and hyperglycemia in the generation of self-sustained limbic seizures. Furthermore, this study suggests the control of glucose availability as a potential therapeutic tool for SE.

The association between chronotypes and season of birth (SOB) remains an inconclusive issue due, in some extension, to the lack of investigations of mediation mechanisms. We evaluated the association of photoperiod at birth (PAB) with chronotypes and sleep duration in Brazil (n = 810), and the mediating effect of meteorological factors, sex, age and rs4753426 polymorphism in the melatonin receptor MTNR1B. Longer PAB was associated with a delayed mid-sleep phase with a suppressive effect of maximum environmental temperature. No significant interactions were identified for the other variables. These findings suggest that photoperiod and environmental temperature modulate chronotype development at early stages.

Crack users suffer the effects of cocaine present in the drug and the action of other active compounds from its pyrolysis. An emergent fact is an increase in the number of pregnant crack cocaine users. Studies suggest that crack cocaine and its metabolites cross the placenta, promoting premature birth, fever, irritability, sweating, and seizures in the early months of life. In children, the effects of crack cocaine have been associated with cognitive deficits, difficulty in verbalization, aggressiveness, and depression, besides enhancing the susceptibility to epileptic seizures, including status epilepticus (SE) in adulthood. Therefore, we investigated the effect of maternal exposure to smoke crack cocaine on several behavioral parameters in the offspring during adulthood. A series of behavioral tests and intrahippocampal pilocarpine (H-PILO) microinjection at sub-convulsive and convulsive doses in a rat model demonstrated that exposure to crack cocaine during the embryonic period leads to anxiogenic-like behavior and long-term memory impairment in both genders and promotes depressive-like behavior in the female. Besides, crack cocaine offspring exposed to a sub-convulsive H-PILO dose showed higher susceptibility to SE, increased seizure frequency, and neurodegeneration, while animals that received a convulsive dose of H-PILO displayed no alteration in SE severity. Taken together, our data suggest that crack cocaine exposure during the gestational period leads to an increased predilection for anxiety and depression, long-term memory deficits, and reduction in the threshold for developing epileptic seizures associated with neuronal death, which predispose crack cocaine babies to develop neuropsychological disorders.

Background Evidences suggest that alterations in circadian rhythms trigger the development of mental disorders. Eveningness, sleep behavior, and circadian genes polymorphisms have been associated with depression and anxiety symptomatology. However, the mechanism underlying these interactions is not well understood. We investigated the contribution of diurnal preference, sleep habits, and PER3 VNTR polymorphism (rs57875989) to depression and anxiety symptoms in a Northeast sample from the Brazilian population. Methods Eight hundred and four young adults completed the Morningness-Eveningness (MEQ), Munich Chronotype (MCTQ), Center for Epidemiologic Studies – Depression (CES-D), and Beck Anxiety Inventory (BAI) questionnaires. All participants were genotyped and linear regression was performed to test the interactions between the genetic /behavioral variants and depression/ anxiety symptoms. Results Eveningness and sleep behaviors (bedtime, wake-up time, sleep duration, and midpoint of sleep) were correlated with depression symptomatology, specifically in somatic factors of the CES-D questionnaire. No correlation was found between diurnal preference/sleep habits with anxiety symptoms for both BAI total score and its factors. However, women with PER34/4 genotype showed less interpesonal affect in depression symptomatology and more anxiety symptoms in four factors of the BAI questionnaire. Limitations Mainly because this study was based on self-report questionnaires and was limited to undergraduate students aging 18 to 30 years old. Conclusion These results reinforce a role for sleep and diurnal preference in depression, and PER3 VNTR polymorphism in anxiety symptomatology, particularly in women.

Grafting of neural stem cells (NSCs) derived from human induced pluripotent stem cells (hiPSCs) has shown promise for brain repair after injury or disease, but safety issues have hindered their clinical application. Employing nano-sized extracellular vesicles (EVs) derived from hiPSC-NSCs appears to be a safer alternative because they likely have similar neuroreparative properties as NSCs and are amenable for non-invasive administration as an autologous or allogeneic off-the-shelf product. However, reliable methods for isolation, characterization and testing the biological properties of EVs are critically needed for translation. We investigated signatures of miRNAs and proteins and the biological activity of EVs, isolated from hiPSC-NSCs through a combination of anion-exchange chromatography (AEC) and size-exclusion chromatography (SEC). AEC and SEC facilitated the isolation of EVs with intact ultrastructure and expressing CD9, CD63, CD81, ALIX and TSG 101. Small RNA sequencing, proteomic analysis, pathway analysis and validation of select miRNAs and proteins revealed that EVs were enriched with miRNAs and proteins involved in neuroprotective, anti-apoptotic, antioxidant, anti-inflammatory, blood-brain barrier repairing, neurogenic and Aβ reducing activities. Besides, EVs comprised miRNAs and/or proteins capable of promoting synaptogenesis, synaptic plasticity and better cognitive function. Investigations using an in vitro macrophage assay and a mouse model of status epilepticus confirmed the anti-inflammatory activity of EVs. Furthermore, the intranasal administration of EVs resulted in the incorporation of EVs by neurons, microglia and astrocytes in virtually all adult rat and mouse brain regions, and enhancement of hippocampal neurogenesis. Thus, biologically active EVs containing miRNAs and proteins relevant to brain repair could be isolated from hiPSC-NSC cultures, making them a suitable biologic for treating neurodegenerative disorders.

An initial precipitating injury in the brain, such as after status epilepticus (SE), evolves into chronic temporal lobe epilepsy (TLE). We investigated changes in the miRNA composition of extracellular vesicles (EVs) in the forebrain after the establishment of SE-induced chronic TLE. We induced SE in young Fischer 344 rats through graded intraperitoneal injections of kainic acid, which resulted in consistent spontaneous recurrent seizures at ~ 3 months post-SE. We isolated EVs from the entire forebrain of chronically epileptic rats and age-matched naive control animals through an ultracentrifugation method and performed miRNA-sequencing studies to discern changes in the miRNA composition of forebrain-derived EVs in chronic epilepsy. EVs from both naive and epileptic forebrains displayed spherical or cup-shaped morphology, a comparable size range, and CD63 expression but lacked the expression of a deep cellular marker GM130. However, miRNA-sequencing studies suggested downregulation of 3 miRNAs (miR-187-5p, miR-346, and miR-331-3p) and upregulation of 4 miRNAs (miR-490-5p, miR-376b-3p, miR-493-5p, and miR-124-5p) in EVs from epileptic forebrains with fold changes ranging from 1.5 to 2.4 (p < 0.0006; FDR < 0.05). By using geNorm and Normfinder software, we identified miR-487 and miR-221 as the best combination of reference genes for measurement of altered miRNAs found in the epileptic forebrain through qRT-PCR studies. The validation revealed that only miR-346 and miR-331-3p were significantly downregulated in EVs from the epileptic forebrain. The enrichment pathway analysis of these miRNAs showed an overrepresentation of signaling pathways that are linked to molecular mechanisms underlying chronic epilepsy, including GABA-ergic (miR-346 targets) and mTOR (miR-331-3p targets) systems. Thus, the packaging of two miRNAs into EVs in neural cells is considerably altered in chronic epilepsy. Functional studies on these two miRNAs may uncover their role in the pathophysiology and treatment of TLE.

Cognitive dysfunction and neuroinflammation are conspicuously observed in Gulf War Illness (GWI). We investigated whether brain inflammation in GWI is associated with activation of high mobility group box-1 (HMGB1) and complement-related proteins in neurons and astrocytes, and brain inflammation can be tracked through neuron-derived extracellular vesicles (NDEVs) and astrocyte-derived EVs (ADEVs) found in the circulating blood. We exposed animals to GWI-related chemicals pyridostigmine bromide, DEET and permethrin, and moderate stress for 28 days. We performed behavioral tests 10 months post-exposure and quantified activated microglia and reactive astrocytes in the cerebral cortex. Then, we measured the concentration of HMGB1, proinflammatory cytokines, and complement activation-related proteins in the cerebral cortex, and NDEVs and ADEVs in the circulating blood. Cognitive impairments persisted in GWI rats at 10 months post-exposure, which were associated with increased density of activated microglia and reactive astrocytes in the cerebral cortex. Moreover, the level of HMGB1 was elevated in the cerebral cortex with altered expression in the cytoplasm of neuronal soma and dendrites as well as the extracellular space. Also, higher levels of proinflammatory cytokines (TNFa, IL-1b, and IL-6), and complement activation-related proteins (C3 and TccC5b-9) were seen in the cerebral cortex. Remarkably, increased levels of HMGB1 and proinflammatory cytokines observed in the cerebral cortex of GWI rats could also be found in NDEVs isolated from the blood. Similarly, elevated levels of complement proteins seen in the cerebral cortex could be found in ADEVs. The results provide new evidence that persistent cognitive dysfunction and chronic neuroinflammation in a model of GWI are linked with elevated HMGB1 concentration and complement activation. Furthermore, the results demonstrated that multiple biomarkers of neuroinflammation could be tracked reliably via analyses of NDEVs and ADEVs in the circulating blood. Execution of such a liquid biopsy approach is especially useful in clinical trials for monitoring the remission, persistence or progression of brain inflammation in GWI patients with drug treatment.

Made available in DSpace on 2021-06-25T12:24:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-11-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) The most common form of genetic generalized epilepsy (GGE) is juvenile myodonic epilepsy (JME), which accounts for 5 to 10% of all epilepsy cases. The gene EFHC1 has been implicated as a putative cause of JME. However, it remains debatable whether testing for EFHC1 mutations should be included in the diagnostic epilepsy gene panels. To investigate the clinical utility of EFHC1 testing, we studied 125 individuals: 100 with JME and 25 with other GGEs. We amplified and sequenced all EFHC1 coding exons. Then, we predicted the pathogenicity or benign impact of the variants using the analyses proposed by the American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP). Mutation screening revealed 11 missense variants in 44 probands with JME (44%) and one of the seven individuals with generalized tonic-clonic seizures on awakening (14%). Six of the 11 variants ( 54%) were classified as 'benign,' and the remaining variants were considered variants of uncertain significance (VUS). There is currently a limitation to test for genes that predispose an individual to complex, nonmonogenic phenotypes. Thus, we show suggestive evidence that EFHC1 testing lacks a scientific foundation based on the disputed nature of the gene-disease relationship and should be currently limited to research purposes. (C) 2020 Elsevier Inc. All rights reserved. Univ Campinas UNICAMP, Sch Med Sci, Dept Med Genet & Genom Med, Tessalia Vieira Camargo 126, BR-13083887 Campinas, SP, Brazil Univ Campinas UNICAMP, Sch Med Sci, Dept Neurol, Campinas, SP, Brazil Brazilian Inst Neurosci & Neurotechnol BRAINN, Campinas, SP, Brazil Fed Univ Alagoas UFAL, Inst Biol Sci & Hlth, Maceio, Alagoas, Brazil Sao Paulo State Univ UNESP, Sch Med, Dept Neurol & Psychiat, Botucatu, SP, Brazil Sao Paulo State Univ UNESP, Sch Med, Dept Neurol & Psychiat, Botucatu, SP, Brazil FAPESP: FAPESP: 2013/07559-3 CNPq: 143189/2009-3 CNPq: 403299/2016-0 CNPq: 309494/2014-1

Temporal lobe epilepsy (TLE) is characterized by spontaneous recurrent seizures, starting from secondary functional disorders due to several insults, including self-sustaining continuous seizures identified as status epilepticus (SE). Although hypoglycemia has been associated with SE, the effect of inhibition of the Na(+)/glucose cotransporters (SGLTs) on hippocampus during SE is still unknown. Here we evaluated the functional role of SGLT in the pattern of limbic seizures and neurodegeneration process after pilocarpine (PILO)-induced SE. Vehicle (VEH, 1μL) or phlorizin, a specific SGLT inhibitor (PZN, 1μL, 50μg/μL), was administered in the hippocampus of rats 30min before PILO (VEH+PILO or PZN+PILO, respectively). The limbic seizures were classified using the Racine's scale, and the amount of wet dog shakes (WDS) was quantified before and during SE. Neurodegeneration process was evaluated by Fluoro-Jade C (FJ-C), and FJ-C-positive neurons (FJ-C+) were counted 24h and 15days after SE. The PZN-treated rats showed higher (p

Extracellular vesicles (EVs) derived from human bone marrow mesenchymal stem cells (hMSCs) have great promise as biologics to treat neurological and neurodegenerative conditions due to their robust antiinflammatory and neuroprotective properties. Besides, intranasal (IN) administration of EVs has caught much attention because the procedure is noninvasive, amenable for repetitive dispensation, and leads to a quick penetration of EVs into multiple regions of the forebrain. Nonetheless, it is unknown whether brain injury-induced signals are essential for the entry of IN-administered EVs into different brain regions. Therefore, in this study, we investigated the distribution of IN-administered hMSC-derived EVs into neurons and microglia in the intact and status epilepticus (SE) injured rat forebrain. Ten billion EVs labeled with PKH26 were dispensed unilaterally into the left nostril of na&iuml, ve rats, and rats that experienced two hours of kainate-induced SE. Six hours later, PKH26 + EVs were quantified from multiple forebrain regions using serial brain sections processed for different neural cell markers and confocal microscopy. Remarkably, EVs were seen bilaterally in virtually all regions of intact and SE-injured forebrain. The percentage of neurons incorporating EVs were comparable for most forebrain regions. However, in animals that underwent SE, a higher percentage of neurons incorporated EVs in the hippocampal CA1 subfield and the entorhinal cortex, the regions that typically display neurodegeneration after SE. In contrast, the incorporation of EVs by microglia was highly comparable in every region of the forebrain measured. Thus, unilateral IN administration of EVs is efficient for delivering EVs bilaterally into neurons and microglia in multiple regions in the intact or injured forebrain. Furthermore, incorporation of EVs by neurons is higher in areas of brain injury, implying that injury-related signals likely play a role in targeting of EVs into neurons, which may be beneficial for EV therapy in various neurodegenerative conditions including traumatic brain injury, stroke, multiple sclerosis, and Alzheimer&rsquo, s disease.

Mesial Temporal Lobe Epilepsy (mTLE) characterized by progressive development of complex partial seizures originating from the hippocampus is the most prevalent and refractory type of epilepsy. One of the remarkable features of mTLE is the rhythmic pattern of occurrence of spontaneous seizures, implying a dependence on the endogenous clock system for seizure threshold. Conversely, circadian rhythms are affected by epilepsy too. Comprehending how the circadian system and seizures interact with each other is essential for understanding the pathophysiology of epilepsy as well as for developing innovative therapies that are efficacious for better seizure control. In this review, we confer how the temporal dysregulation of the circadian clock in the hippocampus combined with multiple uncoupled oscillators could lead to periodic seizure occurrences and comorbidities. Unraveling these associations with additional research would help in developing chronotherapy for mTLE, based on the chronobiology of spontaneous seizures. Notably, differential dosing of antiepileptic drugs over the circadian period and/or strategies that resynchronize biological rhythms may substantially improve the management of seizures in mTLE patients.

Background Melatonin modulates the master circadian clock through the activation of G-protein-coupled receptors MT1 and MT2. It is presumed, therefore, that genetic variations in melatonin receptors can affect both sleep and circadian phase. We investigated whether the −1193T > C (rs4753426) polymorphism in the promoter of MT2 receptor gene (MTNR1B) is associated with diurnal preference and sleep habits. This polymorphism was previously associated with sunshine duration, suggesting a role in circadian entrainment. Methods A total of 814 subjects who completed the Morningness–Eveningness and the Munich Chronotype questionnaires were genotyped for the selected polymorphism. Linear and multinomial regression were performed to test the interaction between gene variants and diurnal preference/sleep habits. Results The −1193C variant was associated with the extreme morningness phenotype in a codominant model (C/C vs. T/T), recessive model (C/C + C/T vs. T/T) and alleles (C vs. T). A negative correlation was found between −1193C alleles and social jetlag scores. The frequency of −1193T allele was higher in the group that stay in bed more than 8 h/night compared to the group that stay in bed less than 8 h/night on weekends. Conclusion To the best of our knowledge, these data provide the first insights into the role of MTNR1B gene in the regulation of sleep, biological rhythms, and entrainment in humans.

Neuropathological studies often use autopsy brain tissue as controls to evaluate changes in protein or RNA levels in several diseases. In mesial temporal lobe epilepsy (MTLE), several genes are up or down regulated throughout the epileptogenic and chronic stages of the disease. Given that postmortem changes in several gene transcripts could impact the detection of changes in case-control studies, we evaluated the effect of using autopsy specimens with different postmortem intervals (PMI) on differential gene expression of the Pilocarpine (PILO)induced Status Epilepticus (SE) of MTLE. For this, we selected six genes (Gfap, Ppia, Gad65, Gad67, Npy, and Tnf-α) whose expression patterns in the hippocampus of PILO-injected rats are well known. Initially, we compared hippocampal expression of naïve rats whose hippocampi were harvested immediately after death (0h-PMI) with those harvested at 6h postmortem interval (6h-PMI): Npy and Ppia transcripts increased and Tnf-α transcripts decreased in the 6h-PMI group (p

Many animal prototypes illustrating the various attributes of human temporal lobe epilepsy (TLE) are available. These models have been invaluable for comprehending multiple epileptogenic processes, modifications in electrophysiological properties, neuronal hyperexcitability, neurodegeneration, neural plasticity, and chronic neuroinflammation in TLE. Some models have also uncovered the efficacy of new antiepileptic drugs or biologics for alleviating epileptogenesis, cognitive impairments, or spontaneous recurrent seizures (SRS). Nonetheless, the suitability of these models for testing candidate therapeutics in conditions such as chronic TLE is debatable because of a lower frequency of SRS and an inconsistent pattern of SRS activity over days, weeks or months. An ideal prototype of chronic TLE for investigating novel therapeutics would need to display a large number of SRS with a dependable frequency and severity and related co-morbidities. This study presents a new kainic acid (KA) model of chronic TLE generated through induction of status epilepticus (SE) in 6-8 weeks old male F344 rats. A rigorous characterization in the chronic epilepsy period validated that the animal prototype mimicked the most salient features of robust chronic TLE. Animals displayed a constant frequency and intensity of SRS across weeks and months in the 5th and 6th month after SE, as well as cognitive and mood impairments. Moreover, SRS frequency displayed a rhythmic pattern with 24-hour periodicity and a consistently higher number of SRS in the daylight period. Besides, the model showed many neuropathological features of chronic TLE, which include a partial loss of inhibitory interneurons, reduced neurogenesis with persistent aberrant migration of newly born neurons, chronic neuroinflammation typified by hypertrophied astrocytes and rod-shaped microglia, and a significant aberrant mossy fiber sprouting in the hippocampus. This consistent chronic seizure model is ideal for investigating the efficacy of various antiepileptic drugs and biologics as well as understanding multiple pathophysiological mechanisms underlying chronic epilepsy.

There is little evidence for the involvement of microRNAs (miRs) in the regulation of circadian rhythms, despite the potential relevance of these elements in the posttranscriptional regulation of the clock machinery. The present work aimed to identify miRs targeting circadian genes through a predictive analysis of conserved miRs in mammals. Besides 23 miRs previously associated with circadian rhythms, we found a number of interesting candidate genes, equally predicted by the 3 software programs used, including miR-9, miR-24, miR25, miR-26, miR-27, miR-29, miR-93, miR-211, miR-302, and miR-346. Moreover, several miRs are predicted to be regulated by circadian transcription factors, such as CLOCK/BMAL, DEC2, and REV-ERBalpha. Using real-time PCR we demonstrated that the selected candidate miR-27b showed a daily variation in human leukocytes. This study presents predicted feedback loops for mammalian molecular clock and the first description of an miR with in vivo daily variation in humans.

The involvement of miRNA in mesial temporal lobe epilepsy (MTLE) pathogenesis has increasingly become a focus of epigenetic studies. Despite advances, the number of known miRNAs with a consistent expression response during epileptogenesis is still small. Addressing this situation requires additional miRNA profiling studies coupled to detailed individual expression analyses. Here, we perform a miRNA microarray analysis of the hippocampus of Wistar rats 24 hours after intra-hippocampal pilocarpine-induced Status Epilepticus (H-PILO SE). We identified 73 miRNAs that undergo significant changes, of which 36 were up-regulated and 37 were down-regulated. To validate, we selected 5 of these (10a-5p, 128a-3p, 196b-5p, 352 and 324-3p) for RT-qPCR analysis. Our results confirmed that miR-352 and 196b-5p levels were significantly higher and miR-128a-3p levels were significantly lower in the hippocampus of H-PILO SE rats. We also evaluated whether the 3 miRNAs show a dysregulated hippocampal expression at three time periods (0h, 24h and chronic phase) after systemic pilocarpine-induced status epilepticus (S-PILO SE). We demonstrate that miR-128a-3p transcripts are significantly reduced at all time points compared to the naive group. Moreover, miR-196b-5p was significantly higher only at 24h post-SE, while miR-352 transcripts were significantly up-regulated after 24h and in chronic phase (epileptic) rats. Finally, when we compared hippocampi of epileptic and non-epileptic humans, we observed that transcript levels of miRNAs show similar trends to the animal models. In summary, we successfully identified two novel dysregulated miRNAs (196b-5p and 352) and confirmed miR-128a-3p downregulation in SE-induced epileptogenesis. Further functional assays are required to understand the role of these miRNAs in MTLE pathogenesis.

The aim of this study was to identify molecular pathways involved in audiogenic seizures in the epilepsy-prone Wistar Audiogenic Rat (WAR). For this, we used a suppression-subtractive hybridization (SSH) library from the hippocampus of WARs coupled to microarray comparative gene expression analysis, followed by Northern blot validation of individual genes. We discovered that the levels of the non-protein coding (npc) RNA BC1 were significantly reduced in the hippocampus of WARs submitted to repeated audiogenic seizures (audiogenic kindling) when compared to Wistar resistant rats and to both naive WARs and Wistars. By quantitative in situ hybridization, we verified lower levels of BC1 RNA in the GD-hilus and significant signal ratio reduction in the stratum radiatum and stratum pyramidale of hippocampal CA3 subfield of audiogenic kindled animals. Functional results recently obtained in a BC1⁻/⁻ mouse model and our current data are supportive of a potential disruption in signaling pathways, upstream of BC1, associated with the seizure susceptibility of WARs.

INTRODUÇÃO: Epilepsia é uma desordem neurológica caracterizada por crises espontâneas e recorrentes, que afeta de 2% a 3 % da população mundial. As crises epilépticas refletem atividade elétrica anormal e paroxística, preferencialmente em uma ou várias áreas do córtex cerebral, que podem ser causadas por inúmeras patologias estruturais ou neuroquímicas. Dentre os importantes estudos das últimas décadas no campo da epileptologia, destaca-se a identificação de genes associados a certos tipos de epilepsia. OBJETIVO: Nesta revisão, descrevemos as principais alterações genéticas associadas ao processo epileptogênico, discutindo as mais recentes descobertas e suas contribuições para a compreensão das bases genéticas das epilepsias idiopáticas monogênicas (EIM) e das epilepsias geneticamente complexas. RESULTADOS E CONCLUSÃO: Estudos de ligação e associação mostram que alterações em genes que codificam canais iônicos são as principais causas genéticas das epilepsias idiopáticas monogênicas e de predisposição nas epilepsias geneticamente complexas. Além disso, as síndromes nas quais a epilepsia é um aspecto importante do quadro clínico podem ser provocadas por genes envolvidos em diferentes vias celulares, tais como: migração neuronal, metabolismo de glicogênio e cadeia respiratória. Portanto, acredita-se que diferentes categorias de genes possam atuar na determinação do traço epiléptico. A identificação de tais famílias de genes não apenas nos ajudará a entender as vias moleculares associadas à hiperexcitabilidade neuronal e ao processo epileptogênico, mas também poderá conduzir ao desenvolvimento de novas e mais precisas estratégias de tratamento da epilepsia. INTRODUCTION: Epilepsy is a neurological disorder characterized by spontaneous and recurrent seizures with an estimated prevalence of 2-3 % in the world population. Epileptic seizures are the result of paroxystic and hypersynchronous electrical activity, preferentially in cortical areas, caused by panoply of structural and neurochemical dysfunctions. Recent advances in the field have focused on the molecular mechanisms involved in the epileptogenic process. OBJECTIVES: In the present review, we describe the main genetic alterations associated to the process of epileptogenesis and discuss the new findings that are shedding light on the molecular substrates of monogenic idiopathic epilepsies (MIE) and on genetically complex epilepsies (GCE). RESULTS AND CONCLUSION: Linkage and association studies have shown that mutations in ion channel genes are the main causes of MIE and of predisposition for GCE. Moreover, mutations in genes involved in neuronal migration, glycogen metabolism and respiratory chain are associated to other syndromes involving seizures. Therefore, different gene classes contribute to the epileptic trait. The identification of epilepsy-related gene families can help us understand the molecular mechanisms of neuronal hyperexcitability and recognize markers of early diagnosis as well as new treatments for these epilepsies.

Epileptic seizures are hypersynchronous, paroxystic and abnormal neuronal discharges. Epilepsies are characterized by diverse mechanisms involving alteration of excitatory and inhibitory neurotransmission that result in hyperexcitability of the central nervous system (CNS). Enhanced neuronal excitability can also be achieved by inflammatory processes, including the participation of cytokines, prostaglandins or kinins, molecules known to be involved in either triggering or in the establishment of inflammation. Multiple inductions of audiogenic seizures in the Wistar audiogenic rat (WAR) strain are a model of temporal lobe epilepsy (TLE), due to the recruitment of limbic areas such as hippocampus and amygdala. In this study we investigated the modulation of the B1 and B2 kinin receptors expression levels in neonatal WARs as well as in adult WARs subjected to the TLE model. The expression levels of pro-inflammatory (IL-1 beta) and anti-inflammatory (IL-10) cytokines were also evaluated, as well as cyclooxygenase (COX-2). Our results showed that the B1 and B2 kinin receptors mRNAs were up-regulated about 7- and 4-fold, respectively, in the hippocampus of kindled WARs. On the other hand, the expressions of the IL-1 beta, IL-10 and COX-2 were not related to the observed increase of expression of kinin receptors. Based on those results we believe that the B1 and B2 kinin receptors have a pivotal role in this model of TLE, although their participation is not related to an inflammatory process. We believe that kinin receptors in the CNS may act in seizure mechanisms by participating in a specific kininergic neurochemical pathway.

Juvenile myoclonic epilepsy (JME) accounts for 26% of generalized idiopathic epileptic syndromes. The highest levels of thrombin activity are closely involved in the development of neurological diseases, including epilepsy. The prothrombin c.20210G>A (rs1799963) variation, which alters prothrombin mRNA stability, is associated with high plasma prothrombin levels. Objective : The present study was designed to investigate whether the SNP rs1799963 is a risk factor for JME in the northeastern Brazilian population. Results : The polymorphism was genotyped in 207 controls and 123 patients using polymerase chain reaction-restriction fragment length polymorphism method. No significant differences were observed in the genotype and allele frequencies of this polymorphism between cases and controls. Conclusion : These results present no evidence for an association of rs1799963 with JME. Further studies including other types of epilepsy are required to investigate the involvement of prothrombin gene in the genetic susceptibility to chronic seizure. Epilepsia mioclônica juvenil (EMJ) representa 26% das síndromes epilépticas idiopáticas generalizadas. Níveis elevados de atividade da trombina estão intimamente envolvidos no desenvolvimento de distúrbios neurológicos, incluindo epilepsia. A variante c.20210G>A (rs1799963) do gene de protrombina, que altera a estabilidade do RNAm, está associada com altos níveis de protrombina no plasma. Objetivo: Investigar se o SNP rs1799963 é um fator de risco para EMJ em uma amostra da população do nordeste brasileiro. Resultados : O polimorfismo foi genotipado em 123 pacientes e 207 controles usando a reação de polimerase em cadeia com restrição de polimorfismo. Não observamos diferença significativa nas frequências alélicas e genotípicas deste polimorfismo, entre as populações de pacientes e controle. Conclusão : Estes resultados não demonstram evidências para uma associação do polimorfismo rs1799963 com EMJ. Estudos posteriores, incluindo outros tipos de epilepsia, são necessários para investigar o envolvimento do gene protrombina na susceptibilidade genética a crises crônicas.

The molecular mechanisms underlying epileptogenesis have been widely investigated by differential gene expression approach, especially RT-qPCR methodology. However, controversial findings highlight the occurrence of unpredictable sources of variance in the experimental designs. Here, we investigated if diurnal rhythms of transcript’s levels may impact on differential gene expression analysis in hippocampus of rats with experimental epilepsy. For this, we have selected six core clock genes (Per1, Per3, Bmal1, Clock, Cry1 and Cry2), whose rhythmic expression pattern in hippocampus had been previously reported. Initially, we identified Tubb2a/Rplp1 and Tubb2a/Ppia as suitable normalizers for circadian studies in hippocampus of rats maintained to 12:12 hour light:dark (LD) cycle. Next, we confirmed the temporal profiling of Per1, Per3, Bmal1, Cry1 and Cry2 mRNA levels in the hippocampus of naive rats by both Acrophase and CircWave statistical tests for circadian analysis. Finally, we showed that temporal differences of sampling can change experimental results for Per1, Per3, Bmal1, Cry1 and Cry2, but not for Clock, which was consistently decreased in rats with epilepsy in all comparison to the naive group. In conclusion, our study demonstrates it is mandatory to consider diurnal oscillations, in order to avoid erroneous conclusions in gene expression analysis in hippocampus of rats with epilepsy. Investigators, therefore, should be aware that genes with circadian expression could be out of phase in different animals of experimental and control groups. Moreover, our results indicate that a sub-expression of Clock may be involved in epileptogenicity, although the functional significance of this remains to be investigated.

Circadian rhythms are controlled by a molecular mechanism that is organized in transcriptional and translational feedback loops of gene expression. Recent studies have been demonstrating the involvement of microRNAs (miRs) in post-transcriptional/translational control of circadian rhythms. In the present study we aimed to analyze the daily variations of miR-16 and miR-181a expression in human leukocytes. These miRs were independently associated with hematopoiesis and circadian rhythms in previous studies using experimental models. Peripheral blood from 6 subjects was sampled in a 24 hour period for expression analysis using quantitative real-time PCR (RT-qPCR). Initially, we evaluated the expression stability of RNU6-2, RNU1A-1, RNU5A-1, SNORD-25, SCARNA-17 and SNORA-73A as candidate genes for normalization of RT-qPCR data. The combination of the four most stable genes (SNORA-73A/SCARNA-17/SNORD-25/RNU6-2) was indicated to provide a better normalization of miRs expressions. The results show a daily variation of miR-181a and miR-16 expression in human leukocytes, suggesting a potential participation of these genes in the modulation of the circadian rhythms present in blood cells.

BackgroundSeveral genetic association investigations have been performed over the last three decades to identify variants underlying Juvenile Myoclonic Epilepsy (JME). Here, we evaluate the accumulating findings and provide an updated perspective of these studies.MethodologyA systematic literature search was conducted using the PubMed, Embase, Scopus, Lilacs, epiGAD, Google Scholar and Sigle up to February 12, 2016. The quality of the included studies was assessed by a score and classified as low and high quality. Beyond outcome measures, information was extracted on the setting for each study, characteristics of population samples and polymorphisms.ResultsFifty studies met eligibility criteria and were used for data extraction. With a single exception, all studies used a candidate gene approach, providing data on 229 polymorphisms in or near 55 different genes. Of variants investigating in independent data sets, only rs2029461 SNP in GRM4, rs3743123 in CX36 and rs3918149 in BRD2 showed a significant association with JME in at least two different background populations. The lack of consistent associations might be due to variations in experimental design and/or limitations of the approach.ConclusionsThus, despite intense research evidence established, specific genetic variants in JME susceptibility remain inconclusive. We discussed several issues that may compromise the quality of the results, including methodological bias, endophenotype and potential involvement of epigenetic factors.Prospero registration numberCRD42016036063.

Sleep disturbance is common in several epilepsy types, such as juvenile myoclonic epilepsy (JME). Genetic background could increase susceptibility to seizure and sleep abnormalities. From this perspective, a susceptibility gene for sleep disturbance or chronotype could contribute to the genetic susceptibility threshold for epilepsy and vice versa. Accordingly, we investigated whether functional clock gene polymorphisms (PER2 111C>G, CLOCK 3111T>C, and PER3 VNTR) might influence the risk for JME. All these polymorphisms have recently been reported to be associated with sleep disturbance, diurnal variation, and neurological diseases. The polymorphisms were genotyped in 97 patients and 212 controls using polymerase chain reaction or restriction fragment length polymorphism methods. No significant differences were observed in the genotypic and allelic frequencies of these polymorphisms between cases and controls even when analyses were restricted to patients that presented a diurnal preferential seizure occurrence. We also tested for interactions between polymorphisms by multifactor dimensionality reduction analysis. None of the combined genotypes differed significantly between the groups. These results present no evidence for an association of these polymorphisms with JME. Further studies including other types of epilepsy and/or other functional polymorphisms are required to investigate the possible relationship between clock genes and the genetic susceptibility to chronic seizure.

Real-time quantitative RT-PCR (qPCR) is one of the most powerful techniques for analyzing miRNA expression because of its sensitivity and specificity. However, in this type of analysis, a suitable normalizer is required to ensure that gene expression is unaffected by the experimental condition. To the best of our knowledge, there are no reported studies that performed a detailed identification and validation of suitable reference genes for miRNA qPCR during the epileptogenic process. Here, using a pilocarpine (PILO) model of mesial temporal lobe epilepsy (MTLE), we investigated five potential reference genes, performing a stability expression analysis using geNorm and NormFinder softwares. As a validation strategy, we used each one of the candidate reference genes to measure PILO-induced changes in microRNA-146a levels, a gene whose expression pattern variation in the PILO injected model is known. Our results indicated U6SnRNA and SnoRNA as the most stable candidate reference genes. By geNorm analysis, the normalization factor should preferably contain at least two of the best candidate reference genes (snoRNA and U6SnRNA). In fact, when normalized using the best combination of reference genes, microRNA-146a transcripts were found to be significantly increased in chronic stage, which is consistent with the pattern reported in different models. Conversely, when reference genes were individually employed for normalization, we failed to detect up-regulation of the microRNA-146a gene in the hippocampus of epileptic rats. The data presented here support that the combination of snoRNA and U6SnRNA was the minimum necessary for an accurate normalization of gene expression at the different stages of epileptogenesis that we tested.

Background: Juvenile myoclonic epilepsy (JME) is an idiopathic generalized epilepsy syndrome with genetic basis and accounts for 10% of all forms of epilepsy. Despite the existence of rare mutations responsible for some familial forms inherited in a Mendelian pattern, the genetics of JME is complex and probably involves multiple genes. Because of widespread distribution in the central nervous system (CNS) and their ability to produce postsynaptic inhibition, GABA (A) receptor subunits (GABRs) encoding genes represent high ranking candidates for epilepsy susceptibility. Aim: This case/control study was designed to investigate whether the rs211037 of the GABRG2 gene is a risk factor for JME in the Brazilian population. Materials and Methods: The polymorphism was genotyped in 98 patients and 130 controls using polymerase chain reaction-restriction fragment length polymorphism method. Descriptive and statistical analyses were performed using SNP stat software. Results: Genotype proportions and allele frequencies for the rs211037 polymorphism of the GABARG2 gene did not differ significantly between the groups, even when the odds ratio was adjusted for clinical variables. Conclusion: These results present no evidence for an association of rs211037 with JME. Further studies are required to investigate the involvement of the GABRG2 gene in the genetic susceptibility to this epileptic syndrome.

It is well recognized that the reference gene in a RT-qPCR should be properly validated to ensure that gene expression is unaffected by the experimental condition. We investigated eight potential reference genes in two different pilocarpine PILO-models of mesial temporal lobe epilepsy (MTLE) performing a stability expression analysis using geNorm, NormFinder and BestKepeer softwares. Then, as a validation strategy, we conducted a relative expression analysis of the Gfap gene. Our results indicate that in the systemic PILO-model Actb, Gapdh, Rplp1, Tubb2a and Polr1a mRNAs were highly stable in hippocampus of rats from all experimental and control groups, whereas Gusb revealed to be the most variable one. In fact, we observed that using Gusb for normalization, the relative mRNA levels of the Gfap gene differed from those obtained with stable genes. On the contrary, in the intrahippocampal PILO-model, all softwares included Gusb as a stable gene, whereas B2m was indicated as the worst candidate gene. The results obtained for the other reference genes were comparable to those observed for the systemic Pilo-model. The validation of these data by the analysis of the relative expression of Gfap showed that the upregulation of the Gfap gene in the hippocampus of rats sacrificed 24 hours after status epilepticus (SE) was undetected only when B2m was used as the normalizer. These findings emphasize that a gene that is stable in one pathology model may not be stable in a different experimental condition related to the same pathology and therefore, the choice of reference genes depends on study design.

Background Core promoters are cis-regulatory modules to which bind the basal transcriptional machinery and which participate in the regulation of transcription initiation. Although core promoters have not been extensively investigated through functional assays in a chromosomal context, the available data suggested that the response of a given core promoter might vary depending on the promoter context. Previous studies suggest that a (-57/+40) fragment constitutes the core promoter of the BhC4-1 gene which is located in DNA puff C4 of the sciarid fly Bradysia hygida. Here we tested this (-57/+40) fragment in distinct regulatory contexts in order to verify if promoter context affects its core promoter activity. Results Consistent with the activity of a core promoter, we showed that in the absence of upstream regulatory sequences the (-57/+40) fragment drives low levels of reporter gene mRNA expression throughout development in transgenic Drosophila. By assaying the (-57/+40) fragment in two distinct regulatory contexts, either downstream of the previously characterized Fbp1 enhancer or downstream of the UAS element, we showed that the BhC4-1 core promoter drives regulated transcription in both the germline and in various tissues throughout development. Furthermore, the use of the BhC4-1 core promoter in a UAS construct significantly reduced salivary gland ectopic expression in third instar larvae, which was previously described to occur in the context of the GAL4/UAS system. Conclusions Our results from functional analysis in transgenic Drosophila show that the BhC4-1 core promoter drives gene expression regardless of the promoter context that was assayed. New insights into the functioning of the GAL4/UAS system in Drosophila were obtained, indicating that the presence of the SV40 sequence in the 3' UTR of a UAS construct does not preclude expression in the germline. Furthermore, our analysis indicated that ectopic salivary gland expression in the GAL4/UAS system does not depend only on sequences present in the GAL4 construct, but can also be affected by the core promoter sequences in the UAS construct. In this context, we propose that the sciarid BhC4-1 core promoter constitutes a valuable core promoter which can be employed in functional assays in insects.

The Wistar Audiogenic Rat (WAR) is an epileptic-prone strain developed by genetic selection from a Wistar progenitor based on the pattern of behavioral response to sound stimulation. Chronic acoustic stimulation protocols of WARs (audiogenic kindling) generate limbic epileptogenesis, confirmed by ictal semiology, amygdale, and hippocampal EEG, accompanied by hippocampal and amygdala cell loss, as well as neurogenesis in the dentate gyrus (DG). In an effort to identify genes involved in molecular mechanisms underlying epileptic process, we used suppression-subtractive hybridization to construct normalized cDNA library enriched for transcripts expressed in the hippocampus of WARs. The most represented gene among the 133 clones sequenced was the ionotropic glutamate receptor subunit II (GluR2), a member of the α-amino-3-hydroxy-5-methyl-4-isoxazoleopropionic acid (AMPA) receptor. Although semiquantitative RT-PCR analysis shows that the hippocampal levels of the GluR2 subunits do not differ between naive WARs and their Wistar counterparts, we observed that the expression of the transcript encoding the splice-variant GluR2-flip is increased in the hippocampus of WARs submitted to both acute and kindled audiogenic seizures. Moreover, using in situ hybridization, we verified upregulation of GluR2-flip mainly in the CA1 region, among the hippocampal subfields of audiogenic kindled WARs. Our findings on differential upregulation of GluR2-flip isoform in the hippocampus of WARs displaying audiogenic seizures is original and agree with and extend previous immunohistochemical for GluR2 data obtained in the Chinese P77PMC audiogenic rat strain, reinforcing the association of limbic AMPA alterations with epileptic seizures. © 2009 Wiley-Liss, Inc.

Nesta revisão, introduzimos abordagens investigativas, assim como discutimos os principais achados de expressão gênica diferencial em tecido epiléptico humano e em modelos experimentais. As alterações observadas no cérebro de indivíduos epilépticos sugerem que eventos moleculares específicos refletem diferentes expressões do quadro fisiopatológico. É possível que diferentes combinações da expressão de genes associados à morte celular, metabolismo de radicais livres, transmissão sináptica, resposta imune e de neurotrofinas reflitam propriedades características de diferentes populações neuronais e gliais, que determinam as distintas respostas de cada área cerebral. A compreensão dessas particularidades moleculares será muito importante para o desenvolvimento de uma estratégia de intervenção visando reduzir neurotoxicidade e disfunções sinápticas que ocorrem durante a epileptogênese e a fase crônica em pacientes epilépticos. We introduce some investigative appnacher and findings on differential gene expression in human epileptic time as well as in animal models of epilepsy. Molecular alterations observed in the epileptic brain suggest that they may disclose different psychopathological stages. It is possible that different gene expression combinations involved in cell death, reactive oxygen metabolism, synaptic transmission and immune response and of neurotrophins reflect distinct functional properties of different neuronal and glial populations, which determine specific brain region responses. Understanding the molecular patterns of gene expression following epileptogenic insults will be of great importance for the development of treatments aiming to reduce neurotoxicity and subtle synaptic dyfunctions present in the early stages as well as during the chronic phase of epilepsy.

The sciarid DNA puff C4 BhC4-1 gene is amplified and transcribed in salivary glands at the end of the larval stage. In transgenic Drosophila, the BhC4-1 promoter drives transcription in prepupal salivary glands and in the ring gland of late embryos. A bioinformatics analysis has identified 162 sequences similar to distinct regions of the BhC4-1 proximal promoter, which are predominantly located either in 5′ or 3′ regions or introns in the Drosophila melanogaster genome. A significant number of the identified sequences are found in the regulatory regions of Drosophila genes that are expressed in the salivary gland. Functional assays in Drosophila reveal that the BhC4-1 proximal promoter contains both a 129 bp (−186/−58) salivary gland enhancer and a 67 bp (−253/−187) ring gland enhancer that drive tissue specific patterns of developmentally regulated gene expression, irrespective of their orientation. genesis 46:43–51, 2008. © 2008 Wiley-Liss, Inc.

There are many different studies that contribute to the global picture of the ethnic heterogeneity in Brazilian populations. These studies use different types of genetic markers and are focused on the comparison of populations at different levels. In some of them, each geographical region is treated as a single homogeneous population, whereas other studies create different subdivisions: political (e.g., pooling populations by State), demographic (e.g., urban and rural), or ethnic (e.g., culture, self-declaration, or skin colour). In this study, we performed an enhanced reassessment of the genetic ancestry of ~ 1,300 Brazilians characterised for 46 autosomal Ancestry Informative Markers (AIMs). In addition, 798 individuals from twelve Brazilian populations representing the five geographical macro-regions of Brazil were newly genotyped, including a Native American community and a rural Amazonian community. Following an increasing North to South gradient, European ancestry was the most prevalent in all urban populations (with values up to 74%). The populations in the North consisted of a significant proportion of Native American ancestry that was about two times higher than the African contribution. Conversely, in the Northeast, Center-West and Southeast, African ancestry was the second most prevalent. At an intrapopulation level, all urban populations were highly admixed, and most of the variation in ancestry proportions was observed between individuals within each population rather than among population. Nevertheless, individuals with a high proportion of Native American ancestry are only found in the samples from Terena and Santa Isabel. Our results allowed us to further refine the genetic landscape of Brazilians while establishing the basis for the effective application of an autosomal AIM panel in forensic casework and clinical association studies within the highly admixed Brazilian populations.