Age-dependent regulation of dendritic spine density and protein expression in Mir324 KO mice.

Dendritic spines are small, dynamic protrusions along the dendrite that comprise more than 90% of excitatory connections in the brain, making them essential sites for neuronal communication. These synaptic sites change throughout the process of development, reducing in density and shifting morphology as synapses are refined. One important class of dendritic spine regulators is microRNA (miRNA), small noncoding RNAs that post-transcriptionally regulate gene expression. Several studies suggest that miRNA-324-5p regulates dendritic spine formation. In addition, we have previously shown that miR-324-5p plays a role in seizure and long-term potentiation, both of which involve dendritic spine changes. In this study, we aimed to characterize the role of miRNA-324-5p in developmental spine regulation by assessing the effect of Mir324 knockout (KO) on dendritic spine density and expression of a subset of dendritic proteins at select developmental time points. We show that miR-324-5p expression is developmentally regulated and peaks at four weeks of age. We demonstrate that loss of miR-324-5p expression leads to differential changes in both target protein expression and spine density at different time points during development, disrupting the pattern of spine density changes and leading to a premature loss of dendritic spines in KO mice, which is compensated later. Our findings indicate that miR-324-5p plays a role in synaptic refinement across development. Additionally, our data illustrate the importance of context in the study of miRNA, as regulation by and/or of miRNA can vary dramatically across development and in disease.

Mir324 knockout regulates the structure of dendritic spines and impairs hippocampal long-term potentiation.

MicroRNAs are an emerging class of synaptic regulators. These small noncoding RNAs post-transcriptionally regulate gene expression, thereby altering neuronal pathways and shaping cell-to-cell communication. Their ability to rapidly alter gene expression and target multiple pathways makes them interesting candidates in the study of synaptic plasticity. Here, we demonstrate that the proconvulsive microRNA miR-324-5p regulates excitatory synapse structure and function in the hippocampus of mice. Both Mir324 knockout (KO) and miR-324-5p antagomir treatment significantly reduce dendritic spine density in the hippocampal CA1 subregion, and Mir324 KO, but not miR-324-5p antagomir treatment, shift dendritic spine morphology, reducing the proportion of thin, "unstable" spines. Western blot and quantitative Real-Time PCR revealed changes in protein and mRNA levels for potassium channels, cytoskeletal components, and synaptic markers, including MAP2 and Kv4.2, which are important for long-term potentiation (LTP). In line with these findings, slice electrophysiology revealed that LTP is severely impaired in Mir324 KO mice, while neurotransmitter release probability remains unchanged. Overall, this study demonstrates that miR-324-5p regulates dendritic spine density, morphology, and plasticity in the hippocampus, potentially via multiple cytoskeletal and synaptic modulators.

Age-Dependent Regulation of Dendritic Spine Density and Protein Expression in Mir324 KO Mice.

Dendritic spines are small, dynamic protrusions along the dendrite that comprise more than 90% of excitatory connections in the brain, making them essential sites for neuronal communication. These synaptic sites change throughout the process of development, reducing in density and shifting morphology as synapses are refined. One important class of dendritic spine regulators is microRNA (miRNA), small-noncoding RNAs that post-transcriptionally regulate gene expression. Several studies suggest that miRNA-324-5p regulates dendritic spine formation. In addition, we have previously shown that miR-324-5p plays a role in seizure and long-term potentiation, both of which involve dendritic spine changes. In this study, we aimed to characterize the role of miRNA-324-5p in developmental spine regulation by assessing the effect of Mir324 knockout (KO) on dendritic spine density and expression of a subset of dendritic proteins at select developmental time points. We show that miR-324-5p expression is developmentally regulated and peaks at 4 weeks of age. We demonstrate that loss of miR-324-5p expression leads to differential changes in both target protein expression and spine density at different time points during development, disrupting the pattern of spine density changes and leading to a premature loss of dendritic spines in KO mice, which is compensated later. Our findings indicate that miR-324-5p plays a role in synaptic refinement across development. Additionally, our data illustrate the importance of context in the study of miRNA, as regulation by and/or of miRNA can vary dramatically across development and in disease.

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.

MiR-324-5p inhibition after intrahippocampal kainic acid-induced status epilepticus does not prevent epileptogenesis in mice.

Background: Acquired epilepsies are caused by an initial brain insult that is followed by epileptogenesis and finally the development of spontaneous recurrent seizures. The mechanisms underlying epileptogenesis are not fully understood. MicroRNAs regulate mRNA translation and stability and are frequently implicated in epilepsy. For example, antagonism of a specific microRNA, miR-324-5p, before brain insult and in a model of chronic epilepsy decreases seizure susceptibility and frequency, respectively. Here, we tested whether antagonism of miR-324-5p during epileptogenesis inhibits the development of epilepsy. Methods: We used the intrahippocampal kainic acid (IHpKa) model to initiate epileptogenesis in male wild type C57BL/6 J mice aged 6-8 weeks. Twenty-four hours after IHpKa, we administered a miR-324-5p or scrambled control antagomir intracerebroventricularly and implanted cortical surface electrodes for EEG monitoring. EEG data was collected for 28 days and analyzed for seizure frequency and duration, interictal spike activity, and EEG power. Brains were collected for histological analysis. Results: Histological analysis of brain tissue showed that IHpKa caused characteristic hippocampal damage in most mice regardless of treatment. Antagomir treatment did not affect latency to, frequency, or duration of spontaneous recurrent seizures or interictal spike activity but did alter the temporal development of frequency band-specific EEG power. Conclusion: These results suggest that miR-324-5p inhibition during epileptogenesis induced by status epilepticus does not convey anti-epileptogenic effects despite having subtle effects on EEG frequency bands. Our results highlight the importance of timing of intervention across epilepsy development and suggest that miR-324-5p may act primarily as a proconvulsant rather than a pro-epileptogenic regulator.

Impact of Raptor and Rictor Deletion on Hippocampal Pathology Following Status Epilepticus.

Neuronal hyperactivation of the mTOR signaling pathway may play a role in driving the pathological sequelae that follow status epilepticus. Animal studies using pharmacological tools provide support for this hypothesis, however, systemic inhibition of mTOR-a growth pathway active in every mammalian cell-limits conclusions on cell type specificity. To circumvent the limitations of pharmacological approaches, we developed a viral/genetic strategy to delete Raptor or Rictor, inhibiting mTORC1 or mTORC2, respectively, from excitatory hippocampal neurons after status epilepticus in mice. Raptor or Rictor was deleted from roughly 25% of hippocampal granule cells, with variable involvement of other hippocampal neurons, after pilocarpine status epilepticus. Status epilepticus induced the expected loss of hilar neurons, sprouting of granule cell mossy fiber axons and reduced c-Fos activation. Gene deletion did not prevent these changes, although Raptor loss reduced the density of c-Fos-positive granule cells overall relative to Rictor groups. Findings demonstrate that mTOR signaling can be effectively modulated with this approach and further reveal that blocking mTOR signaling in a minority (25%) of granule cells is not sufficient to alter key measures of status epilepticus-induced pathology. The approach is suitable for producing higher deletion rates, and altering the timing of deletion, which may lead to different outcomes.

Increases in sensory noise predict attentional disruptions to audiovisual speech perception.

We receive information about the world around us from multiple senses which combine in a process known as multisensory integration. Multisensory integration has been shown to be dependent on attention; however, the neural mechanisms underlying this effect are poorly understood. The current study investigates whether changes in sensory noise explain the effect of attention on multisensory integration and whether attentional modulations to multisensory integration occur via modality-specific mechanisms. A task based on the McGurk Illusion was used to measure multisensory integration while attention was manipulated via a concurrent auditory or visual task. Sensory noise was measured within modality based on variability in unisensory performance and was used to predict attentional changes to McGurk perception. Consistent with previous studies, reports of the McGurk illusion decreased when accompanied with a secondary task; however, this effect was stronger for the secondary visual (as opposed to auditory) task. While auditory noise was not influenced by either secondary task, visual noise increased with the addition of the secondary visual task specifically. Interestingly, visual noise accounted for significant variability in attentional disruptions to the McGurk illusion. Overall, these results strongly suggest that sensory noise may underlie attentional alterations to multisensory integration in a modality-specific manner. Future studies are needed to determine whether this finding generalizes to other types of multisensory integration and attentional manipulations. This line of research may inform future studies of attentional alterations to sensory processing in neurological disorders, such as Schizophrenia, Autism, and ADHD.

GABAA Alpha 2,3 Modulation Improves Select Phenotypes in a Mouse Model of Fragile X Syndrome.

Fragile X syndrome (FXS) is the most common cause of inherited intellectual disability. FXS is caused by functional loss of the Fragile X Protein (FXP), also known as Fragile X Mental Retardation Protein (FMRP). In humans and animal models, loss of FXP leads to sensory hypersensitivity, increased susceptibility to seizures and cortical hyperactivity. Several components of the GABAergic system, the major inhibitory system in the brain, are dysregulated in FXS, and thus modulation of GABAergic transmission was suggested and tested as a treatment strategy. However, so far, clinical trials using broad spectrum GABAA or GABAB receptor-specific agonists have not yielded broad improvement of FXS phenotypes in humans. Here, we tested a more selective strategy in Fmr1 knockout (KO) mice using the experimental drug BAER-101, which is a selective GABAA α2/α3 agonist. Our results suggest that BAER-101 reduces hyperexcitability of cortical circuits, partially corrects increased frequency-specific baseline cortical EEG power, reduces susceptibility to audiogenic seizures and improves novel object memory. Other Fmr1 KO-specific phenotypes were not improved by the drug, such as increased hippocampal dendritic spine density, open field activity and marble burying. Overall, this work shows that BAER-101 improves select phenotypes in Fmr1 KO mice and encourages further studies into the efficacy of GABAA-receptor subunit-selective agonists for the treatment of FXS.

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.