Investigation of MicroRNA-134 as a Target against Seizures and SUDEP in a Mouse Model of Dravet Syndrome

Dravet syndrome (DS) is a catastrophic form of pediatric epilepsy mainly caused by noninherited mutations in the SCN1A gene. DS patients suffer severe and life-threatening focal and generalized seizures which are often refractory to available anti-seizure medication. Antisense oligonucleotides (ASOs) based approaches may offer treatment opportunities in DS. MicroRNAs are short noncoding RNAs that play a key role in brain structure and function by post-transcriptionally regulating gene expression, including ion channels. Inhibiting miRNA-134 (miR-134) using an antimiR ASO (Ant-134) has been shown to reduce evoked seizures in juvenile and adult mice and reduce epilepsy development in models of focal epilepsy. The present study investigated the levels of miR-134 and whether Ant-134 could protect against hyperthermia-induced seizures, spontaneous seizures and mortality (SUDEP) in F1.Scn1a(1/)tm1kea mice. At P17, animals were intracerebroventricular in-jected with 0.1–1 nmol of Ant-134 and subject to a hyperthermia challenge at postnatal day (P)18. A second cohort of P21 F1.Scn1a(1/)tm1kea mice received Ant-134 and were followed by video and EEG monitoring until P28 to track the incidence of spontaneous seizures and SUDEP. Hippocampal and cortical levels of miR-134 were similar between wild-type (WT) and F1.Scn1a(1/)tm1kea mice. Moreover, Ant-134 had no effect on hyperthermia-induced seizures, spontaneous seizures and SUDEP incidence were unchanged in Ant-134-treated DS mice. These findings suggest that targeting miR-134 does not have therapeutic applications in DS

Spared CA1 pyramidal neuron function and hippocampal performance following antisense knockdown of microRNA-134

OBJECTIVE: Inhibition of microRNA-134 by an oligonucleotide antagomir (ant-134) has been shown to produce powerful antiseizure effects in multiple models of epilepsy. However, to successfully translate the treatment to the clinic, it is important to assess what potential adverse effects it may have on naive brain tissue. METHODS: To investigate this, adult male Sprague-Dawley rats were treated with either ant-134 or a scrambled control sequence. Animals were later assessed for spatial navigation, before ex vivo slices were taken to assess the effects of microRNA-134 knockdown on well-defined measures of intrinsic and synaptic properties. RESULTS: Hippocampal field potential recordings determined that silencing of microRNA-134 by ant-134 injection was associated with a reduction in epileptiform activity following application of 9 mmol/L K+ . Nevertheless, rats performed normally in the novel object location test. Action potential waveforms and miniature excitatory synaptic currents recorded in CA1 pyramidal neurons were unaffected by ant-134. SIGNIFICANCE: These results demonstrate that ant-134 confers a seizure-protective effect without obvious interference with hippocampal neuronal properties or network function. These findings support further development of this novel approach to epilepsy treatment

Antagomir-mediated suppression of microRNA-134 reduces kainic acid-induced seizures in immature mice

MicroRNAs are short non-coding RNAs that negatively regulate protein levels and perform important roles in establishing and maintaining neuronal network function. Previous studies in adult rodents have detected upregulation of microRNA-134 after prolonged seizures (status epilepticus) and demonstrated that silencing microRNA-134 using antisense oligonucleotides, termed antagomirs, has potent and long-lasting seizure-suppressive effects. Here we investigated whether targeting microRNA-134 can reduce or delay acute seizures in the immature brain. Status epilepticus was induced in 21 day-old (P21) male mice by systemic injection of 5 mg/kg kainic acid. This triggered prolonged electrographic seizures and select bilateral neuronal death within the CA3 subfield of the hippocampus. Expression of microRNA-134 and functional loading to Argonaute-2 was not significantly changed in the hippocampus after seizures in the model. Nevertheless, when levels of microRNA-134 were reduced by prior intracerebroventricular injection of an antagomir, kainic acid-induced seizures were delayed and less severe and mice displayed reduced neuronal death in the hippocampus. These studies demonstrate targeting microRNA-134 may have therapeutic applications for the treatment of seizures in children

Reduced Mature MicroRNA Levels in Association with Dicer Loss in Human Temporal Lobe Epilepsy with Hippocampal Sclerosis

Hippocampal sclerosis (HS) is a common pathological finding in patients with temporal lobe epilepsy (TLE) and is associated with altered expression of genes controlling neuronal excitability, glial function, neuroinflammation and cell death. MicroRNAs (miRNAs), a class of small non-coding RNAs, function as post-transcriptional regulators of gene expression and are critical for normal brain development and function. Production of mature miRNAs requires Dicer, an RNAase III, loss of which has been shown to cause neuronal and glial dysfunction, seizures, and neurodegeneration. Here we investigated miRNA biogenesis in hippocampal and neocortical resection specimens from pharmacoresistant TLE patients and autopsy controls. Western blot analysis revealed protein levels of Dicer were significantly lower in certain TLE patients with HS. Dicer levels were also reduced in the hippocampus of mice subject to experimentally-induced epilepsy. To determine if Dicer loss was associated with altered miRNA processing, we profiled levels of 380 mature miRNAs in control and TLE-HS samples. Expression of nearly 200 miRNAs was detected in control human hippocampus. In TLE-HS samples there was a large-scale reduction of miRNA expression, with 51% expressed at lower levels and a further 24% not detectable. Primary transcript (pri-miRNAs) expression levels for several tested miRNAs were not different between control and TLE-HS samples. These findings suggest loss of Dicer and failure of mature miRNA expression may be a feature of the pathophysiology of HS in patients with TLE

Dissection of a QTL Hotspot on Mouse Distal Chromosome 1 that Modulates Neurobehavioral Phenotypes and Gene Expression

A remarkably diverse set of traits maps to a region on mouse distal chromosome 1 (Chr 1) that corresponds to human Chr 1q21–q23. This region is highly enriched in quantitative trait loci (QTLs) that control neural and behavioral phenotypes, including motor behavior, escape latency, emotionality, seizure susceptibility (Szs1), and responses to ethanol, caffeine, pentobarbital, and haloperidol. This region also controls the expression of a remarkably large number of genes, including genes that are associated with some of the classical traits that map to distal Chr 1 (e.g., seizure susceptibility). Here, we ask whether this QTL-rich region on Chr 1 (Qrr1) consists of a single master locus or a mixture of linked, but functionally unrelated, QTLs. To answer this question and to evaluate candidate genes, we generated and analyzed several gene expression, haplotype, and sequence datasets. We exploited six complementary mouse crosses, and combed through 18 expression datasets to determine class membership of genes modulated by Qrr1. Qrr1 can be broadly divided into a proximal part (Qrr1p) and a distal part (Qrr1d), each associated with the expression of distinct subsets of genes. Qrr1d controls RNA metabolism and protein synthesis, including the expression of ∼20 aminoacyl-tRNA synthetases. Qrr1d contains a tRNA cluster, and this is a functionally pertinent candidate for the tRNA synthetases. Rgs7 and Fmn2 are other strong candidates in Qrr1d. FMN2 protein has pronounced expression in neurons, including in the dendrites, and deletion of Fmn2 had a strong effect on the expression of few genes modulated by Qrr1d. Our analysis revealed a highly complex gene expression regulatory interval in Qrr1, composed of multiple loci modulating the expression of functionally cognate sets of genes

Cellular injury and neuroinflammation in children with chronic intractable epilepsy

<p>Abstract</p> <p>Objective</p> <p>To elucidate the presence and potential involvement of brain inflammation and cell death in neurological morbidity and intractable seizures in childhood epilepsy, we quantified cell death, astrocyte proliferation, microglial activation and cytokine release in brain tissue from patients who underwent epilepsy surgery.</p> <p>Methods</p> <p>Cortical tissue was collected from thirteen patients with intractable epilepsy due to focal cortical dysplasia (6), encephalomalacia (5), Rasmussen's encephalitis (1) or mesial temporal lobe epilepsy (1). Sections were processed for immunohistochemistry using markers for neuron, astrocyte, microglia or cellular injury. Cytokine assay was performed on frozen cortices. Controls were autopsy brains from eight patients without history of neurological diseases.</p> <p>Results</p> <p>Marked activation of microglia and astrocytes and diffuse cell death were observed in epileptogenic tissue. Numerous fibrillary astrocytes and their processes covered the entire cortex and converged on to blood vessels, neurons and microglia. An overwhelming number of neurons and astrocytes showed DNA fragmentation and its magnitude significantly correlated with seizure frequency. Majority of our patients with abundant cell death in the cortex have mental retardation. IL-1beta, IL-8, IL-12p70 and MIP-1beta were significantly increased in the epileptogenic cortex; IL-6 and MCP-1 were significantly higher in patients with family history of epilepsy.</p> <p>Conclusions</p> <p>Our results suggest that active neuroinflammation and marked cellular injury occur in pediatric epilepsy and may play a common pathogenic role or consequences in childhood epilepsy of diverse etiologies. Our findings support the concept that immunomodulation targeting activated microglia and astrocytes may be a novel therapeutic strategy to reduce neurological morbidity and prevent intractable epilepsy.</p

Microglia Are Mediators of Borrelia burgdorferi–Induced Apoptosis in SH-SY5Y Neuronal Cells

Inflammation has long been implicated as a contributor to pathogenesis in many CNS illnesses, including Lyme neuroborreliosis. Borrelia burgdorferi is the spirochete that causes Lyme disease and it is known to potently induce the production of inflammatory mediators in a variety of cells. In experiments where B. burgdorferi was co-cultured in vitro with primary microglia, we observed robust expression and release of IL-6 and IL-8, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP-1β) and CCL5 (RANTES), but we detected no induction of microglial apoptosis. In contrast, SH-SY5Y (SY) neuroblastoma cells co-cultured with B. burgdorferi expressed negligible amounts of inflammatory mediators and also remained resistant to apoptosis. When SY cells were co-cultured with microglia and B. burgdorferi, significant neuronal apoptosis consistently occurred. Confocal microscopy imaging of these cell cultures stained for apoptosis and with cell type-specific markers confirmed that it was predominantly the SY cells that were dying. Microarray analysis demonstrated an intense microglia-mediated inflammatory response to B. burgdorferi including up-regulation in gene transcripts for TLR-2 and NFκβ. Surprisingly, a pathway that exhibited profound changes in regard to inflammatory signaling was triggering receptor expressed on myeloid cells-1 (TREM1). Significant transcript alterations in essential p53 pathway genes also occurred in SY cells cultured in the presence of microglia and B. burgdorferi, which indicated a shift from cell survival to preparation for apoptosis when compared to SY cells cultured in the presence of B. burgdorferi alone. Taken together, these findings indicate that B. burgdorferi is not directly toxic to SY cells; rather, these cells become distressed and die in the inflammatory surroundings generated by microglia through a bystander effect. If, as we hypothesized, neuronal apoptosis is the key pathogenic event in Lyme neuroborreliosis, then targeting microglial responses may be a significant therapeutic approach for the treatment of this form of Lyme disease

Neuronal Deletion of Caspase 8 Protects against Brain Injury in Mouse Models of Controlled Cortical Impact and Kainic Acid-Induced Excitotoxicity

system. mice demonstrated superior survival, reduced seizure severity, less apoptosis, and reduced caspase 3 processing. Uninjured aged knockout mice showed improved learning and memory, implicating a possible role for caspase 8 in cognitive decline with aging.Neuron-specific deletion of caspase 8 reduces brain damage and improves post-traumatic functional outcomes, suggesting an important role for this caspase in pathophysiology of acute brain trauma

Transcriptome Analysis of the Hippocampal CA1 Pyramidal Cell Region after Kainic Acid-Induced Status Epilepticus in Juvenile Rats

Molecular mechanisms involved in epileptogenesis in the developing brain remain poorly understood. The gene array approach could reveal some of the factors involved by allowing the identification of a broad scale of genes altered by seizures. In this study we used microarray analysis to reveal the gene expression profile of the laser microdissected hippocampal CA1 subregion one week after kainic acid (KA)-induced status epilepticus (SE) in 21-day-old rats, which are developmentally roughly comparable to juvenile children. The gene expression analysis with the Chipster software generated a total of 1592 differently expressed genes in the CA1 subregion of KA-treated rats compared to control rats. The KEGG database revealed that the identified genes were involved in pathways such as oxidative phosporylation (26 genes changed), and long-term potentiation (LTP; 18 genes changed). Also genes involved in Ca2+ homeostasis, gliosis, inflammation, and GABAergic transmission were altered. To validate the microarray results we further examined the protein expression for a subset of selected genes, glial fibrillary protein (GFAP), apolipoprotein E (apo E), cannabinoid type 1 receptor (CB1), Purkinje cell protein 4 (PEP-19), and interleukin 8 receptor (CXCR1), with immunohistochemistry, which confirmed the transcriptome results. Our results showed that SE resulted in no obvious CA1 neuronal loss, and alterations in the expression pattern of several genes during the early epileptogenic phase were comparable to previous gene expression studies of the adult hippocampus of both experimental epileptic animals and patients with temporal lobe epilepsy (TLE). However, some changes seem to occur after SE specifically in the juvenile rat hippocampus. Insight of the SE-induced alterations in gene expression and their related pathways could give us hints for the development of new target-specific antiepileptic drugs that interfere with the progression of the disease in the juvenile age group