Estradiol- and Progesterone-Associated Changes in microRNA-Induced Silencing and Reduced Antiseizure Efficacy of an Antagomir in Female Mice.

About one-third of individuals living with epilepsy have treatment-resistant seizures. Alternative therapeutic strategies are thus urgently needed. One potential novel treatment target is miRNA-induced silencing, which is differentially regulated in epilepsy. Inhibitors (antagomirs) of specific microRNAs (miRNAs) have shown therapeutic promise in preclinical epilepsy studies; however, these studies were mainly conducted in male rodent models, and research into miRNA regulation in females and by female hormones in epilepsy is scarce. This is problematic because female sex and the menstrual cycle can affect the disease course of epilepsy and may, therefore, also alter the efficacy of potential miRNA-targeted treatments. Here, we used the proconvulsant miRNA miR-324-5p and its target, the potassium channel Kv4.2, as an example to test how miRNA-induced silencing and the efficacy of antagomirs in epilepsy are altered in female mice. We showed that Kv4.2 protein is reduced after seizures in female mice similar to male mice; however, in contrast to male mice, miRNA-induced silencing of Kv4.2 is unchanged, and miR-324-5p activity, as measured by the association with the RNA-induced silencing complex, is reduced in females after seizure. Moreover, an miR-324-5p antagomir does not consistently reduce seizure frequency or increase Kv4.2 in female mice. As a possible underlying mechanism, we found that miR-324-5p activity and the silencing of Kv4.2 in the brain were differentially correlated with plasma levels of 17ß-estradiol and progesterone. Our results suggest that hormonal fluctuations in sexually mature female mice influence miRNA-induced silencing and could alter the efficacy of potential future miRNA-based treatments for epilepsy in females.

A nerve injury-specific long noncoding RNA promotes neuropathic pain by increasing Ccl2 expression.

Maladaptive changes of nerve injury-associated genes in dorsal root ganglia (DRGs) are critical for neuropathic pain genesis. Emerging evidence supports the role of long noncoding RNAs (lncRNAs) in regulating gene transcription. Here we identified a conserved lncRNA, named nerve injury-specific lncRNA (NIS-lncRNA) for its upregulation in injured DRGs exclusively in response to nerve injury. This upregulation was triggered by nerve injury-induced increase in DRG ELF1, a transcription factor that bound to the NIS-lncRNA promoter. Blocking this upregulation attenuated nerve injury-induced CCL2 increase in injured DRGs and nociceptive hypersensitivity during the development and maintenance periods of neuropathic pain. Mimicking NIS-lncRNA upregulation elevated CCL2 expression, increased CCL2-mediated excitability in DRG neurons, and produced neuropathic pain symptoms. Mechanistically, NIS-lncRNA recruited more binding of the RNA-interacting protein FUS to the Ccl2 promoter and augmented Ccl2 transcription in injured DRGs. Thus, NIS-lncRNA participates in neuropathic pain likely by promoting FUS-triggered DRG Ccl2 expression and may be a potential target in neuropathic pain management.

Cannabinoid Receptor 1 Expression in Human Dorsal Root Ganglia and CB13-Induced Bidirectional Modulation of Sensory Neuron Activity.

Cannabinoid receptors have been identified as potential targets for analgesia from studies on animal physiology and behavior, and from human clinical trials. Here, we sought to improve translational understanding of the mechanisms of cannabinoid-mediated peripheral analgesia. Human lumbar dorsal root ganglia were rapidly recovered from organ donors to perform physiological and anatomical investigations into the potential for cannabinoids to mediate analgesia at the level of the peripheral nervous system. Anatomical characterization of in situ gene expression and immunoreactivity showed that 61 and 53% of human sensory neurons express the CB1 gene and receptor, respectively. Calcium influx evoked by the algogen capsaicin was measured by Fura-2AM in dissociated human sensory neurons pre-exposed to the inflammatory mediator prostaglandin E2 (PGE2) alone or together with CB13 (1 µM), a cannabinoid agonist with limited blood-brain barrier permeability. Both a higher proportion of neurons and a greater magnitude of response to capsaicin were observed after exposure to CB13, indicating cannabinoid-mediated sensitization. In contrast, membrane properties measured by patch-clamp electrophysiology demonstrated that CB13 suppressed excitability and reduced action potential discharge in PGE2-pre-incubated sensory neurons, suggesting the suppression of sensitization. This bidirectional modulation of sensory neuron activity suggests that cannabinoids may suppress overall membrane excitability while simultaneously enhancing responsivity to TRPV1-mediated stimuli. We conclude that peripherally restricted cannabinoids may have both pro- and anti-nociceptive effects in human sensory neurons.

The potassium channel Kv4.2 regulates dendritic spine morphology, electroencephalographic characteristics and seizure susceptibility in mice.

The voltage-gated potassium channel Kv4.2 is a critical regulator of dendritic excitability in the hippocampus and is crucial for dendritic signal integration. Kv4.2 mRNA and protein expression as well as function are reduced in several genetic and pharmacologically induced rodent models of epilepsy and autism. It is not known, however, whether reduced Kv4.2 is just an epiphenomenon or a disease-contributing cause of neuronal hyperexcitability and behavioral impairments in these neurological disorders. To address this question, we used male and female mice heterozygous for a Kv.2 deletion and adult-onset manipulation of hippocampal Kv4.2 expression in male mice to assess the role of Kv4.2 in regulating neuronal network excitability, morphology and anxiety-related behaviors. We observed a reduction in dendritic spine density and reduced proportions of thin and stubby spines but no changes in anxiety, overall activity, or retention of conditioned freezing memory in Kv4.2 heterozygous mice compared with wildtype littermates. Using EEG analyses, we showed elevated theta power and increased spike frequency in Kv4.2 heterozygous mice under basal conditions. In addition, the latency to onset of kainic acid-induced seizures was significantly shortened in Kv4.2 heterozygous mice compared with wildtype littermates, which was accompanied by a significant increase in theta power. By contrast, overexpressing Kv4.2 in wildtype mice through intrahippocampal injection of Kv4.2-expressing lentivirus delayed seizure onset and reduced EEG power. These results suggest that Kv4.2 is an important regulator of neuronal network excitability and dendritic spine morphology, but not anxiety-related behaviors. In the future, manipulation of Kv4.2 expression could be used to alter seizure susceptibility in epilepsy.

MicroRNA inhibition upregulates hippocampal A-type potassium current and reduces seizure frequency in a mouse model of epilepsy.

Epilepsy is often associated with altered expression or function of ion channels. One example of such a channelopathy is the reduction of A-type potassium currents in the hippocampal CA1 region. The underlying mechanisms of reduced A-type channel function in epilepsy are unclear. Here, we show that inhibiting a single microRNA, miR-324-5p, which targets the pore-forming A-type potassium channel subunit Kv4.2, selectively increased A-type potassium currents in hippocampal CA1 pyramidal neurons in mice. Resting membrane potential, input resistance and other potassium currents were not altered. In a mouse model of acquired chronic epilepsy, inhibition of miR-324-5p reduced the frequency of spontaneous seizures and interictal epileptiform spikes supporting the physiological relevance of miR-324-5p-mediated control of A-type currents in regulating neuronal excitability. Mechanistic analyses demonstrated that microRNA-induced silencing of Kv4.2 mRNA is increased in epileptic mice leading to reduced Kv4.2 protein levels, which is mitigated by miR-324-5p inhibition. By contrast, other targets of miR-324-5p were unchanged. These results suggest a selective miR-324-5p-dependent mechanism in epilepsy regulating potassium channel function, hyperexcitability and seizures.