•Hyperactive mTOR signaling is implicated in several seizure-induced brain changes.•Neuroinflammation can impact hyperexcitability and promote epileptogenesis.•mTOR and inflammatory signaling ...interact in both physiological and diseased states.•Dual inhibition of both processes could be a potential anti-epileptogenic therapy.
Existing therapies for epilepsy are primarily symptomatic and target mechanisms of neuronal transmission in order to restore the excitatory/inhibitory imbalance in the brain after seizures. However, approximately one third of individuals with epilepsy have medically refractory epilepsy and do not respond to available treatments. There is a critical need for the development of therapeutics that extend beyond manipulation of excitatory neurotransmission and target pathological changes underlying the cause of the disease. Epilepsy is a multifaceted condition, and it could be that effective treatment involves the targeting of several mechanisms. There is evidence for both dysregulated PI3K/Akt/mTOR (mTOR) signaling and heightened neuroinflammatory processes following seizures in the brain. Signaling via mTOR has been implicated in several epileptogenic processes, including synaptic plasticity mechanisms and changes in ion channel expression following seizures. Inflammatory signaling, such as increased synthesis of cytokines and other immune molecules, has also shown to play a significant role in the development of chronic epilepsy. mTOR pathway activation and immune signaling are known to interact in normal physiological states, as well as influence one another following seizures. Simultaneous inhibition of both processes could be a promising therapeutic avenue to prevent the development of chronic epilepsy by targeting two key pathological mechanisms implicated in epileptogenesis.
•Wnt/β-catenin pathway is disrupted in acute and chronic epileptic phases.•Wnt/β-catenin signaling can regulate seizure-induced neurogenesis.•Multiple mechanisms exist to modulate Wnt/β-catenin ...signaling after seizures.•Pathway is a potential anti-epileptogenic target for future epilepsy therapies.
Epilepsy is one of the most common neurological disorders, and yet many afflicted individuals are resistant to all available therapeutic treatments. Existing pharmaceutical treatments function primarily to reduce hyperexcitability and prevent seizures, but fail to influence the underlying pathophysiology of the disorder. Recently, research efforts have focused on identifying alternative mechanistic targets for anti-epileptogenic therapies that can prevent the development of chronic epilepsy. The Wnt/β-catenin pathway, one possible target, has been demonstrated to be disrupted in both acute and chronic phases of epilepsy. Wnt/β-catenin signaling can regulate many seizure-induced changes in the brain, including neurogenesis and neuronal death, as well as can influence seizure susceptibility and potentially the development of chronic epilepsy. Several genome-wide studies and in vivo knockout animal models have provided evidence for an association between disrupted Wnt/β-catenin signaling and epilepsy. Furthermore, approved pharmaceutical drugs and other small molecule compounds that target components of the β-catenin destruction complex or antagonize endogenous inhibitors of the pathway have shown to be protective following seizures. However, additional studies are needed to determine the optimal time period in which modulation of the pathway may be most beneficial. Overall, disrupted molecular networks such as Wnt/β-catenin signaling, could be a promising anti-epileptogenic target for future epilepsy therapies.
The current study aimed to further address important questions regarding the therapeutic efficacy of omega-3 fatty acids for various behavioral and neuroimmune aspects of the Fmr1 phenotype. To ...address these questions, our experimental design utilized two different omega-3 fatty acid administration timepoints, compared to both standard laboratory chow controls ("Standard") and a diet controlling for the increase in fat content ("Control Fat"). In the first paradigm, post-weaning supplementation (after postnatal day 21) with the omega-3 fatty acid diet ("Omega-3") reversed deficits in startle threshold, but not deficits in prepulse inhibition, and the effect on startle threshold was not specific to the Omega-3 diet. However, post-weaning supplementation with both experimental diets also impaired acquisition of a fear response, recall of the fear memory and contextual fear conditioning compared to the Standard diet. The post-weaning Omega-3 diet reduced hippocampal expression of IL-6 and this reduction of IL-6 was significantly associated with diminished performance in the fear conditioning task. In the perinatal experimental paradigm, the Omega-3 diet attenuated hyperactivity and acquisition of a fear response. Additionally, perinatal exposure to the Control Fat diet (similar to a "Western" diet) further diminished nonsocial anxiety in the Fmr1 knockout. This study provides significant evidence that dietary fatty acids throughout the lifespan can significantly impact the behavioral and neuroimmune phenotype of the Fmr1 knockout model.
There have been several reports that individuals with Fragile X syndrome (FXS) and animal models of FXS have communication deficits. The present study utilized two different call classification ...taxonomies to examine the sex‐specificity of ultrasonic vocalization (USV) production on postnatal day (PD8) in the FVB strain of Fmr1 knockout (KO) mice. One classification protocol requires the investigator to score each call by hand, while the other protocol uses an automated algorithm. Results using the hand‐scoring protocol indicated that male Fmr1 KO mice exhibited longer calls (P = .03) than wild types on PD8. Male KOs also produced fewer complex, composite, downward, short and two‐syllable call‐types, as well as more frequency steps and chevron call‐types. Female heterozygotes exhibited no significant changes in acoustic or temporal aspects of calls, yet showed significant changes in call‐type production proportions across two different classification taxonomies (P < .001). They exhibited increased production of harmonic and frequency steps calls, as well as fewer chevron, downward and short calls. According to the second high‐throughput analysis, female heterozygotes produced significantly fewer single‐type and more multiple‐type syllables, unlike male KOs that showed no changes in these aspects of syllable production. Finally, we correlated both scoring methods and found a high level of correlation between the two methods. These results contribute further knowledge of sex differences in USV calling behavior for Fmr1 heterozygote and KO mice and provide a foundation for the use of high‐throughput analysis of neonatal USVs.
Male Fmr1 KOs demonstrate quantitative changes in vocalizations on day 8, while female Fmr1 HETs show only qualitative changes.
•NS-Pten KO mice display both quantitative and qualitative changes in USVs.•NS-Pten KO mice emit USVs of a lower peak frequency than WT mice.•NS-Pten KO’s emit USVs of a higher amplitude and shorter ...duration than WT mice.•Spectral analysis reveal NS-Pten KO mice produce different types of calls.
The NS-Pten knockout (KO) mouse exhibits hyperactivity of the mammalian target of rapamycin (mTOR) and is a model of autism spectrum disorder (ASD). ASD presents with marked deficits in communication which can be elucidated by investigating their counterpart in mice, ultrasonic vocalizations (USVs). While USVs have been found to be altered in NS-Pten KO pups, no study has assessed whether this communication deficit persists into adulthood. In the present study, we investigate female urine-induced USVs, scent marking behavior, and open field activity in NS-Pten KO and wildtype (WT) adult male mice. Results showed that there was no difference in the quantity of vocalizations produced between groups, however, there were extensive alterations in the spectral properties of USVs. KO mice emitted vocalizations of a lower peak frequency, shorter duration, and higher peak amplitude compared to WT mice. KO animals also emitted a significantly different distribution of call-types relative to controls, displaying increased complex and short calls, but fewer upward, chevron, frequency steps, and composite calls. No significant differences between groups were observed for scent marking behavior and there was no difference between groups in the amount of time spent near the female urine. Overall, this study demonstrated that mTOR hyperactivity contributes to communication deficits in adult mice.
Dravet syndrome is an intractable developmental and epileptic encephalopathy caused by de novo variants in SCN1A resulting in haploinsufficiency of the voltage-gated sodium channel Nav1.1. We showed ...previously that administration of the antisense oligonucleotide STK-001, also called ASO-22, generated using targeted augmentation of nuclear gene output technology to prevent inclusion of the nonsense-mediated decay, or poison, exon 20N in human SCN1A, increased productive Scn1a transcript and Nav1.1 expression and reduced the incidence of electrographic seizures and sudden unexpected death in epilepsy in a mouse model of Dravet syndrome. Here, we investigated the mechanism of action of ASO-84, a surrogate for ASO-22 that also targets splicing of SCN1A exon 20N, in Scn1a+/- Dravet syndrome mouse brain. Scn1a +/- Dravet syndrome and wild-type mice received a single intracerebroventricular injection of antisense oligonucleotide or vehicle at postnatal Day 2. We examined the electrophysiological properties of cortical pyramidal neurons and parvalbumin-positive fast-spiking interneurons in brain slices at postnatal Days 21-25 and measured sodium currents in parvalbumin-positive interneurons acutely dissociated from postnatal Day 21-25 brain slices. We show that, in untreated Dravet syndrome mice, intrinsic cortical pyramidal neuron excitability was unchanged while cortical parvalbumin-positive interneurons showed biphasic excitability with initial hyperexcitability followed by hypoexcitability and depolarization block. Dravet syndrome parvalbumin-positive interneuron sodium current density was decreased compared to wild-type. GABAergic signalling to cortical pyramidal neurons was reduced in Dravet syndrome mice, suggesting decreased GABA release from interneurons. ASO-84 treatment restored action potential firing, sodium current density and GABAergic signalling in Dravet syndrome parvalbumin-positive interneurons. Our work suggests that interneuron excitability is selectively affected by ASO-84. This new work provides critical insights into the mechanism of action of this antisense oligonucleotide and supports the potential of antisense oligonucleotide-mediated upregulation of Nav1.1 as a successful strategy to treat Dravet syndrome.
The voltage-gated Na+ channel β1 subunit, encoded by SCN1B, regulates cell surface expression and gating of α subunits and participates in cell adhesion. β1 is cleaved by α/β and γ-secretases, ...releasing an extracellular domain and intracellular domain (ICD), respectively. Abnormal SCN1B expression/function is linked to pathologies including epilepsy, cardiac arrhythmia, and cancer. In this study, we sought to determine the effect of secretase cleavage on β1 function in breast cancer cells. Using a series of GFP-tagged β1 constructs, we show that β1-GFP is mainly retained intracellularly, particularly in the endoplasmic reticulum and endolysosomal pathway, and accumulates in the nucleus. Reduction in endosomal β1-GFP levels occurred following γ-secretase inhibition, implicating endosomes and/or the preceding plasma membrane as important sites for secretase processing. Using live-cell imaging, we also report β1ICD-GFP accumulation in the nucleus. Furthermore, β1-GFP and β1ICD-GFP both increased Na+ current, whereas β1STOP-GFP, which lacks the ICD, did not, thus highlighting that the β1-ICD is necessary and sufficient to increase Na+ current measured at the plasma membrane. Importantly, although the endogenous Na+ current expressed in MDA-MB-231 cells is tetrodotoxin (TTX)-resistant (carried by Nav1.5), the Na+ current increased by β1-GFP or β1ICD-GFP was TTX-sensitive. Finally, we found β1-GFP increased mRNA levels of the TTX-sensitive α subunits SCN1A/Nav1.1 and SCN9A/Nav1.7. Taken together, this work suggests that the β1-ICD is a critical regulator of α subunit function in cancer cells. Our data further highlight that γ-secretase may play a key role in regulating β1 function in breast cancer.
•No difference in average quantity of calls produced between Fmr1 KO and WT mice.•Fmr1 KO mice emit vocalizations at a higher frequency and decreased amplitude.•Fmr1 KO vocalizations were of ...significantly shorter duration.•Spectral analysis reveal Fmr1 KO mice to produce different types of calls.•Results emphasize qualitative differences in Fmr1 KO mice vocalizations.
The Fmr1 knockout (KO) mouse has commonly been used to investigate communication impairments, one of the key diagnostic symptoms observed in Fragile X syndrome (FXS) and Autism spectrum disorder (ASD). Many studies have found alterations in ultrasonic vocalizations (USVs) in neonatal Fmr1 KO mice, however, there is limited research investigating whether these deficits continue into adulthood. In the present study, we examine differences in female urine-induced ultrasonic vocalizations, scent marking behavior, odor discrimination, and open field activity in adult male Fmr1 KO and wildtype (WT) mice. Overall, we found extensive alterations between genotypes in both spectral and temporal properties of ultrasonic vocalizations. There was no difference in the average number of calls emitted by both genotypes, however, Fmr1 KO mice emitted calls of a higher frequency, decreased amplitude, and shorter duration than WT mice. Spectrographic analyses revealed statistically significant differences between genotypes in the types of calls emitted. Contrastingly, we found no differences in scent marking behavior, a form of social communication, or in odor discrimination and activity levels of the mice. The results corroborate previous studies emphasizing the importance of qualitative differences observed in vocalization behavior of Fmr1 KO mice, rather than quantitative measurements such as number of calls emitted. Overall, the study confirms the presence of abnormalities in vocalization behavior in adult Fmr1 KO mice that we believe are consistent with communication deficits seen in the syndrome.
Using high-throughput analysis methods, the present study sought to determine the impact of prenatal high-fat dietary manipulations on isolation-induced ultrasonic vocalization production in both ...male and female Fmr1mutants on postnatal day 9. Prior to breeding, male FVB/129 Fmr1 wildtype and female Fmr1 heterozygous breeding pairs were assigned to 1 of 3 diet conditions: standard lab chow, omega-3 fatty acid-enriched chow, and a diet controlling for the fat increase. Prenatal exposure to omega-3 fatty acids improved reductions in the number of calls produced by Fmr1heterozygotes females. Moreover, diminished spectral purity in the female Fmr1homozygous mouse was rescued by exposure to both high-fat diets, although these effects were not seen in the male Fmr1knockout. Prenatal dietary fat manipulation also influenced several other aspects of vocalization production, such as the number of calls produced and their fundamental frequency, aside from effects due to loss of Fmr1.Specifically, in males, regardless of genotype, prenatal exposure to high omega-3s increased the average fundamental frequency of calls. These data support the need for future preclinical and clinical work elucidating the full potential of prenatal high-fat diets as a novel therapeutic alternative forFragile X syndrome.
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