Neuronal network activity and connectivity are impaired in a conditional knockout mouse model with PCDH19 mosaic expression.

Mutations in PCDH19 gene, which encodes protocadherin-19 (PCDH19), cause Developmental and Epileptic Encephalopathy 9 (DEE9). Heterogeneous loss of PCDH19 expression in neurons is considered a key determinant of the disorder; however, how PCDH19 mosaic expression affects neuronal network activity and circuits is largely unclear. Here, we show that the hippocampus of Pcdh19 mosaic mice is characterized by structural and functional synaptic defects and by the presence of PCDH19-negative hyperexcitable neurons. Furthermore, global reduction of network firing rate and increased neuronal synchronization have been observed in different limbic system areas. Finally, network activity analysis in freely behaving mice revealed a decrease in excitatory/inhibitory ratio and functional hyperconnectivity within the limbic system of Pcdh19 mosaic mice. Altogether, these results indicate that altered PCDH19 expression profoundly affects circuit wiring and functioning, and provide new key to interpret DEE9 pathogenesis.

Electronic nicotine delivery systems (ENDS): A convenient means of smoking?

Electronic nicotine delivery systems (ENDS), which are becoming increasingly popular in many parts of the world, have recently become more sophisticated in terms of their more active content and better controlled vaporisation. This review begins by describing how cigarette smoking led to the development of ENDS as a means of combatting nicotine addiction. ENDS are usually categorised as belonging to one of only three main generations, but a fourth has been added in order to differentiate the latest, most powerful, most advanced and innovative that have improved heating efficiency. Descriptions of the principal substances contained in ENDS are followed by considerations concerning the risk of toxicity due to the presence of albeit low concentrations of such a variety of compounds inhaled over a long time, and the increasingly widespread use of ENDS as a means of smoking illicit drugs. We also review the most widely used pharmacotherapeutic approaches to smoking cessation, and recent epidemiological data showing that ENDS can help some people to stop smoking. However, in order to ensure their appropriate regulation, there is a need for higher-quality evidence concerning the health effects and safety of ENDS, and their effectiveness in discouraging tobacco smoking.

Developmental impaired Akt signaling in the Shank1 and Shank3 double knock-out mice.

Human mutations and haploinsufficiency of the SHANK family genes are associated with autism spectrum disorders (ASD) and intellectual disability (ID). Complex phenotypes have been also described in all mouse models of Shank mutations and deletions, consistent with the heterogeneity of the human phenotypes. However, the specific role of Shank proteins in synapse and neuronal functions remain to be elucidated. Here, we generated a new mouse model to investigate how simultaneously deletion of Shank1 and Shank3 affects brain development and behavior in mice. Shank1-Shank3 DKO mice showed a low survival rate, a developmental strong reduction in the activation of intracellular signaling pathways involving Akt, S6, ERK1/2, and eEF2 during development and a severe behavioral impairments. Our study suggests that Shank1 and Shank3 proteins are essential to developmentally regulate the activation of Akt and correlated intracellular pathways crucial for mammalian postnatal brain development and synaptic plasticity. Therefore, Akt function might represent a new therapeutic target for enhancing cognitive abilities of syndromic ASD patients.

LRRK2 G2019S kinase activity triggers neurotoxic NSF aggregation.

Parkinson's disease is characterized by the progressive degeneration of dopaminergic neurons within the substantia nigra pars compacta and the presence of protein aggregates in surviving neurons. The LRRK2 G2019S mutation is one of the major determinants of familial Parkinson's disease cases and leads to late-onset Parkinson's disease with pleomorphic pathology, including α-synuclein accumulation and deposition of protein inclusions. We demonstrated that LRRK2 phosphorylates N-ethylmaleimide sensitive factor (NSF). We observed aggregates containing NSF in basal ganglia specimens from patients with Parkinson's disease carrying the G2019S variant, and in cellular and animal models expressing the LRRK2 G2019S variant. We found that LRRK2 G2019S kinase activity induces the accumulation of NSF in toxic aggregates. Of note, the induction of autophagy cleared NSF aggregation and rescued motor and cognitive impairment observed in aged hG2019S bacterial artificial chromosome (BAC) mice. We suggest that LRRK2 G2019S pathological phosphorylation impacts on NSF biochemical properties, thus causing the formation of cytotoxic protein inclusions.

Lateral habenula dysfunctions in Tm4sf2-/y mice model for neurodevelopmental disorder.

Mutations in the TM4SF2 gene, which encodes TSPAN7, cause a severe form of intellectual disability (ID) often comorbid with autism spectrum disorder (ASD). Recently, we found that TM4SF2 loss in mice affects cognition. Here, we report that Tm4sf2-/y mice, beyond an ID-like phenotype, display altered sociability, increased repetitive behaviors, anhedonic- and depressive-like states. Cognition relies on the integration of information from several brain areas. In this context, the lateral habenula (LHb) is strategically positioned to coordinate the brain regions involved in higher cognitive functions. Furthermore, in Tm4sf2-/y mice we found that LHb neurons present hypoexcitability, aberrant neuronal firing pattern and altered sodium and potassium voltage-gated ion channels function. Interestingly, we also found a reduced expression of voltage-gated sodium channel and a hyperactivity of the PKC-ERK pathway, a well-known modulator of ion channels activity, which might explain the functional phenotype showed by Tm4sf2-/y mice LHb neurons. These findings support Tm4sf2-/y mice as useful in modeling some ASD-like symptoms. Additionally, we can speculate that LHb functional alteration in Tm4sf2-/y mice might play a role in the disease pathophysiology.

Altered mRNA Levels of Stress-Related Peptides in Mouse Hippocampus and Caudate-Putamen in Withdrawal after Long-Term Intermittent Exposure to Tobacco Smoke or Electronic Cigarette Vapour.

Nicotine addiction is a severe public health problem. The aim of this study was to investigate the alterations in key neurotransmissions after 60 days of withdrawal from seven weeks of intermittent cigarette smoke, e-cigarette vapours, or an e-cigarette vehicle. In the nicotine withdrawal groups, increased depressive and anxiety/obsessive-compulsive-like behaviours were demonstrated in the tail suspension, sucrose preference and marble burying tests. Cognitive impairments were detected in the spatial object recognition test. A significant increase in Corticotropin-releasing factor (Crf) and Crf1 mRNA levels was observed, specifically after cigarette withdrawal in the caudate-putamen nucleus (CPu). The nociceptin precursor levels were reduced by cigarette (80%) and e-cigarette (50%) withdrawal in the CPu. The delta opioid receptor showed a significant reduction in the hippocampus driven by the exposure to an e-cigarette solubilisation vehicle, while the mRNA levels doubled in the CPu of mice that had been exposed to e-cigarettes. Withdrawal after exposure to e-cigarette vapour induced a 35% Bdnf mRNA decrease in the hippocampus, whereas Bdnf was augmented by 118% by cigarette withdrawal in the CPu. This study shows that long-term withdrawal-induced affective and cognitive symptoms associated to lasting molecular alterations in peptidergic signalling may determine the impaired neuroplasticity in the hippocampal and striatal circuitry.

Persistent cognitive and affective alterations at late withdrawal stages after long-term intermittent exposure to tobacco smoke or electronic cigarette vapour: Behavioural changes and their neurochemical correlates.

Smoking cessation induces a withdrawal syndrome associated with anxiety, depression, and impaired neurocognitive functions, but much less is known about the withdrawal of e-cigarettes (e-CIG). We investigated in Balb/c mice the behavioural and neurochemical effects of withdrawal for up to 90 days after seven weeks' intermittent exposure to e-CIG vapour or cigarette smoke (CIG). The withdrawal of e-CIG and CIG induced early behavioural alterations such as spatial memory deficits (spatial object recognition task), increased anxiety (elevated plus maze test) and compulsive-like behaviour (marble burying test) that persisted for 60-90 days. Notably, attention-related (virtual object recognition task) and depression-like behaviours (tail suspension and sucrose preference tests) appeared only 15-30 days after withdrawal and persisted for as long as up to 90 days. At hippocampal level, the withdrawal-induced changes in the levels of AMPA receptor GluA1 and GluA2/3 subunits, PSD 95 protein, corticotropin-releasing factor (Crf) and Crf receptor 1 (CrfR1) mRNA were biphasic: AMPA receptor subunit and PSD95 protein levels initially remained unchanged and decreased after 60-90 days, whereas Crf/CrfR1 mRNA levels initially increased and then markedly decreased after 60 days. These late reductions correlated with the behavioural impairments, particularly the appearance of depression-like behaviours. Our findings show that major behavioural and neurochemical alterations persist or even first appear late after the withdrawal of chronic CIG smoke or e-CIG vapour exposure, and underline importance of conducting similar studies of humans, including e-CIG vapers.

Impaired approach to novelty and striatal alterations in the oxytocin receptor deficient mouse model of autism.

Long-standing studies established a role for the oxytocin system in social behavior, social reward, pair bonding and affiliation. Oxytocin receptors, implicated in pathological conditions affecting the social sphere such as autism spectrum disorders, can also modulate cognitive processes, an aspect generally overlooked. Here we examined the effect of acute (pharmacological) or genetic (Oxtr-/-) inactivation of oxytocin receptor-mediated signaling, in male mice, in several cognitive tests. In the novel object recognition test, both oxytocin receptor antagonist treated wild type animals and Oxtr-/- mice lacked the typical preference for novelty. Oxtr-/- mice even preferred the familiar object; moreover, their performance in the Morris water maze did not differ from wild types, suggesting that oxytocin receptor inactivation did not disrupt learning. Because the preference for novel objects could be rescued in Oxtr-/- mice with longer habituation periods, we propose that the loss of novelty preferences following Oxtr inactivation is due to altered processing of novel contextual information. Finally, we observed an increased expression of excitatory synaptic markers in the striatum of Oxtr-/- mice and a greater arborization and higher number of spines/neuron in the dorsolateral area of this structure, which drives habit formation. Our data also indicate a specific reshaping of dorsolateral striatal spines in Oxtr-/- mice after exposure to a novel environment, which might subtend their altered approach to novelty, and support previous work pointing at this structure as an important substrate for autistic behaviors.

LSD1 modulates stress-evoked transcription of immediate early genes and emotional behavior.

Behavioral changes in response to stressful stimuli can be controlled via adaptive epigenetic changes in neuronal gene expression. Here we indicate a role for the transcriptional corepressor Lysine-Specific Demethylase 1 (LSD1) and its dominant-negative splicing isoform neuroLSD1, in the modulation of emotional behavior. In mouse hippocampus, we show that LSD1 and neuroLSD1 can interact with transcription factor serum response factor (SRF) and set the chromatin state of SRF-targeted genes early growth response 1 (egr1) and c-fos Deletion or reduction of neuro LSD1 in mutant mice translates into decreased levels of activating histone marks at egr1 and c-fos promoters, dampening their psychosocial stress-induced transcription and resulting in low anxiety-like behavior. Administration of suberoylanilide hydroxamine to neuroLSD1(KO)mice reactivates egr1 and c-fos transcription and restores the behavioral phenotype. These findings indicate that LSD1 is a molecular transducer of stressful stimuli as well as a stress-response modifier. Indeed, LSD1 expression itself is increased acutely at both the transcriptional and splicing levels by psychosocial stress, suggesting that LSD1 is involved in the adaptive response to stress.

The X-Linked Intellectual Disability Protein IL1RAPL1 Regulates Dendrite Complexity.

Mutations and deletions of the interleukin-1 receptor accessory protein like 1 (IL1RAPL1) gene, located on the X chromosome, are associated with intellectual disability (ID) and autism spectrum disorder (ASD). IL1RAPL1 protein is located at the postsynaptic compartment of excitatory synapses and plays a role in synapse formation and stabilization. Here, using primary neuronal cultures and Il1rapl1-KO mice, we characterized the role of IL1RAPL1 in regulating dendrite morphology. In Il1rapl1-KO mice we identified an increased number of dendrite branching points in CA1 and CA2 hippocampal neurons associated to hippocampal cognitive impairment. Similarly, induced pluripotent stem cell-derived neurons from a patient carrying a null mutation of the IL1RAPL1 gene had more dendrites. In hippocampal neurons, the overexpression of full-length IL1RAPL1 and mutants lacking part of C-terminal domains leads to simplified neuronal arborization. This effect is abolished when we overexpressed mutants lacking part of N-terminal domains, indicating that the IL1RAPL1 extracellular domain is required for regulating dendrite development. We also demonstrate that PTPδ interaction is not required for this activity, while IL1RAPL1 mediates the activity of IL-1ß on dendrite morphology. Our data reveal a novel specific function for IL1RAPL1 in regulating dendrite morphology that can help clarify how changes in IL1RAPL1-regulated pathways can lead to cognitive disorders in humans.SIGNIFICANCE STATEMENT Abnormalities in the architecture of dendrites have been observed in a variety of neurodevelopmental, neurodegenerative, and neuropsychiatric disorders. Here we show that the X-linked intellectual disability protein interleukin-1 receptor accessory protein like 1 (IL1RAPL1) regulates dendrite morphology of mice hippocampal neurons and induced pluripotent stem cell-derived neurons from a patient carrying a null mutation of IL1RAPL1 gene. We also found that the extracellular domain of IL1RAPL1 is required for this effect, independently of the interaction with PTPδ, but IL1RAPL1 mediates the activity of IL-1ß on dendrite morphology. Our data reveal a novel specific function for IL1RAPL1 in regulating dendrite morphology that can help clarify how changes in IL1RAPL1-regulated pathways can lead to cognitive disorders in humans.

Pharmacological Modulation of AMPAR Rescues Intellectual Disability-Like Phenotype in Tm4sf2-/y Mice.

Intellectual disability affects 2-3% of the world's population and typically begins during childhood, causing impairments in social skills and cognitive abilities. Mutations in the TM4SF2 gene, which encodes the TSPAN7 protein, cause a severe form of intellectual disability, and currently, no therapy is able to ameliorate this cognitive impairment. We previously reported that, in cultured neurons, shRNA-mediated down-regulation of TSPAN7 affects AMPAR trafficking by enhancing PICK1-GluA2 interaction, thereby increasing the intracellular retention of AMPAR. Here, we found that loss of TSPAN7 function in mice causes alterations in hippocampal excitatory synapse structure and functionality as well as cognitive impairment. These changes occurred along with alterations in AMPAR expression levels. We also found that interfering with PICK1-GluA2 binding restored synaptic function in Tm4sf2-/y mice. Moreover, potentiation of AMPAR activity via the administration of the ampakine CX516 reverted the neurological phenotype observed in Tm4sf2-/y mice, suggesting that pharmacological modulation of AMPAR may represent a new approach for treating patients affected by TM4SF2 mutations and intellectual disability.

eEF2K/eEF2 Pathway Controls the Excitation/Inhibition Balance and Susceptibility to Epileptic Seizures.

Alterations in the balance of inhibitory and excitatory synaptic transmission have been implicated in the pathogenesis of neurological disorders such as epilepsy. Eukaryotic elongation factor 2 kinase (eEF2K) is a highly regulated, ubiquitous kinase involved in the control of protein translation. Here, we show that eEF2K activity negatively regulates GABAergic synaptic transmission. Indeed, loss of eEF2K increases GABAergic synaptic transmission by upregulating the presynaptic protein Synapsin 2b and α5-containing GABAA receptors and thus interferes with the excitation/inhibition balance. This cellular phenotype is accompanied by an increased resistance to epilepsy and an impairment of only a specific hippocampal-dependent fear conditioning. From a clinical perspective, our results identify eEF2K as a potential novel target for antiepileptic drugs, since pharmacological and genetic inhibition of eEF2K can revert the epileptic phenotype in a mouse model of human epilepsy.

Eps8 controls dendritic spine density and synaptic plasticity through its actin-capping activity.

Actin-based remodelling underlies spine structural changes occurring during synaptic plasticity, the process that constantly reshapes the circuitry of the adult brain in response to external stimuli, leading to learning and memory formation. A positive correlation exists between spine shape and synaptic strength and, consistently, abnormalities in spine number and morphology have been described in a number of neurological disorders. In the present study, we demonstrate that the actin-regulating protein, Eps8, is recruited to the spine head during chemically induced long-term potentiation in culture and that inhibition of its actin-capping activity impairs spine enlargement and plasticity. Accordingly, mice lacking Eps8 display immature spines, which are unable to undergo potentiation, and are impaired in cognitive functions. Additionally, we found that reduction in the levels of Eps8 occurs in brains of patients affected by autism compared to controls. Our data reveal the key role of Eps8 actin-capping activity in spine morphogenesis and plasticity and indicate that reductions in actin-capping proteins may characterize forms of intellectual disabilities associated with spine defects.

LSD1 Neurospecific Alternative Splicing Controls Neuronal Excitability in Mouse Models of Epilepsy.

Alternative splicing in the brain is dynamic and instrumental to adaptive changes in response to stimuli. Lysine-specific demethylase 1 (LSD1/KDM1A) is a ubiquitously expressed histone H3Lys4 demethylase that acts as a transcriptional co-repressor in complex with its molecular partners CoREST and HDAC1/2. In mammalian brain, alternative splicing of LSD1 mini-exon E8a gives rise to neuroLSD1, a neurospecific isoform that, upon phosphorylation, acts as a dominant-negative causing disassembly of the co-repressor complex and de-repression of target genes. Here we show that the LSD1/neuroLSD1 ratio changes in response to neuronal activation and such effect is mediated by neurospecific splicing factors NOVA1 and nSR100/SRRM4 together with a novel cis-silencer. Indeed, we found that, in response to epileptogenic stimuli, downregulation of NOVA1 reduces exon E8a splicing and expression of neuroLSD1. Using behavioral and EEG analyses we observed that neuroLSD1-specific null mice are hypoexcitable and display decreased seizure susceptibility. Conversely, in a mouse model of Rett syndrome characterized by hyperexcitability, we measured higher levels of NOVA1 protein and upregulation of neuroLSD1. In conclusion, we propose that, in the brain, correct ratio between LSD1 and neuroLSD1 contributes to excitability and, when altered, could represent a pathogenic event associated with neurological disorders involving altered E/I.

Epileptiform activity and cognitive deficits in SNAP-25(+/-) mice are normalized by antiepileptic drugs.

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a protein that participates in the regulation of synaptic vesicle exocytosis through the formation of the soluble NSF attachment protein receptor complex and modulates voltage-gated calcium channels activity. The Snap25 gene has been associated with schizophrenia, attention deficit hyperactivity disorder, and bipolar disorder, and lower levels of SNAP-25 have been described in patients with schizophrenia. We used SNAP-25 heterozygous (SNAP-25(+/-)) mice to investigate at which extent the reduction of the protein levels affects neuronal network function and mouse behavior. As interactions of genotype with the specific laboratory conditions may impact behavioral results, the study was performed through a multilaboratory study in which behavioral tests were replicated in at least 2 of 3 distinct European laboratories. Reductions of SNAP-25 levels were associated with a moderate hyperactivity, which disappeared in the adult animals, and with impaired associative learning and memory. Electroencephalographic recordings revealed the occurrence of frequent spikes, suggesting a diffuse network hyperexcitability. Consistently, SNAP-25(+/-) mice displayed higher susceptibility to kainate-induced seizures, paralleled by degeneration of hilar neurons. Notably, both EEG profile and cognitive defects were improved by antiepileptic drugs. These results indicate that reduction of SNAP-25 expression is associated to generation of epileptiform discharges and cognitive dysfunctions, which can be effectively treated by antiepileptic drugs.

Cytoarchitectural, behavioural and neurophysiological dysfunctions in the BCNU-treated rat model of cortical dysplasia.

Cortical dysplasias (CDs) include a spectrum of cerebral lesions resulting from cortical development abnormalities during embryogenesis that lead to cognitive disabilities and epilepsy. The experimental model of CD obtained by means of in utero administration of BCNU (1-3-bis-chloroethyl-nitrosurea) to pregnant rats on embryonic day 15 mimics the histopathological abnormalities observed in many patients. The aim of this study was to investigate the behavioural, electrophysiological and anatomical profile of BCNU-treated rats in order to determine whether cortical and hippocampal lesions can directly lead to cognitive dysfunction. The BCNU-treated rats showed impaired short-term working memory but intact long-term aversive memory, whereas their spontaneous motor activity and anxiety-like response were normal. The histopathological and immunohistochemical analyses, made after behavioural tests, revealed the disrupted integrity of neuronal populations and connecting fibres in hippocampus and prefrontal and entorhinal cortices, which are involved in memory processes. An electrophysiological evaluation of the CA1 region of in vitro hippocampal slices indicated a decrease in the efficiency of excitatory synaptic transmission and impaired paired pulse facilitation, but enhanced long-term potentiation (LTP) associated with hyperexcitability in BCNU-treated rats compared with controls. The enhanced LTP, associated with hyperexcitability, may indicate a pathological distortion of long-term plasticity. These findings suggest that prenatal developmental insults at the time of peak cortical neurogenesis can induce anatomical abnormalities associated with severe impairment of spatial working memory in adult BCNU-treated rats and may help to clarify the pathophysiological mechanisms of cognitive dysfunction that is often associated with epilepsy in patients with CD.

Fluoxetine rescues the depressive-like behaviour induced by reserpine and the altered emotional behaviour induced by nicotine withdrawal in zebrafish: Involvement of tyrosine hydroxylase.

BACKGROUND: Nicotine cessation leads to anxiety and depression. AIMS: The suitability of the zebrafish model of anhedonia using reserpine and fluoxetine was evaluated. Fluoxetine was also used to reduce nicotine withdrawal-induced anhedonic state. METHODS: Zebrafish were exposed to reserpine (40 mg/l) and then to fluoxetine (0.1 mg/l) for 1 week. Anhedonia was evaluated in the Novel Tank Diving and Compartment Preference tests. Another group was exposed to nicotine (1 mg/l/2 weeks) and then exposed to fluoxetine. Anxiety and anhedonia were evaluated 2-60 days after. Tyrosine hydroxylase (TH) immunoreactivity and microglial morphology (labelled by 4C4 monoclonal antibody) in the parvocellular pretectal nucleus (PPN), dorsal part, and of calcitonin gene-related peptide (CGRP) in the hypothalamus were also analysed. RESULTS: Less time in the top and increased latency to the top in reserpine compared to a drug-free group was found. Fluoxetine rescued reserpine-induced the reduced time in the top. Seven and 30 days after nicotine withdrawal more time in the bottom and similar time in the Compartment Preference test, rescued by fluoxetine, were shown. In the PPN, 30-day withdrawal induced an increase in TH immunoreactivity, but fluoxetine induced a further significant increase. No changes in PPN microglia morphology and hypothalamic CGRP were detected. CONCLUSIONS: Our findings validate the suitability of the zebrafish model of anhedonia using the reserpine-induced depression-like behaviour and the predictivity using fluoxetine. Fluoxetine rescued nicotine withdrawal-induced anhedonic state, opening the possibility to screen new drugs to alleviate anxiety and depression in smokers during abstinence.

Rescuing epileptic and behavioral alterations in a Dravet syndrome mouse model by inhibiting eukaryotic elongation factor 2 kinase (eEF2K).

BACKGROUND: Dravet Syndrome is a severe childhood pharmaco-resistant epileptic disorder mainly caused by mutations in the SCN1A gene, which encodes for the α1 subunit of the type I voltage-gated sodium channel (NaV1.1), that causes imbalance between excitation and inhibition in the brain. We recently found that eEF2K knock out mice displayed enhanced GABAergic transmission and tonic inhibition and were less susceptible to epileptic seizures. Thus, we investigated the effect of inhibition of eEF2K on the epileptic and behavioral phenotype of Scn1a ± mice, a murine model of Dravet Syndrome. METHODS: To elucidate the role of eEF2K pathway in the etiopathology of Dravet syndrome we generated a new mouse model deleting the eEF2K gene in Scn1a ± mice. By crossing Scn1a ± mice with eEF2K-/- mice we obtained the three main genotypes needed for our studies, Scn1a+/+ eEF2K+/+ (WT mice), Scn1a ± eEF2K+/+ mice (Scn1a ± mice) and Scn1a ± eEF2K-/- mice, that were fully characterized for EEG and behavioral phenotype. Furthermore, we tested the ability of a pharmacological inhibitor of eEF2K in rescuing EEG alterations of the Scn1a ± mice. RESULTS: We showed that the activity of eEF2K/eEF2 pathway was enhanced in Scn1a ± mice. Then, we demonstrated that both genetic deletion and pharmacological inhibition of eEF2K were sufficient to ameliorate the epileptic phenotype of Scn1a ± mice. Interestingly we also found that motor coordination defect, memory impairments, and stereotyped behavior of the Scn1a ± mice were reverted by eEF2K deletion. The analysis of spontaneous inhibitory postsynaptic currents (sIPSCs) suggested that the rescue of the pathological phenotype was driven by the potentiation of GABAergic synapses. LIMITATIONS: Even if we found that eEF2K deletion was able to increase inhibitory synapses function, the molecular mechanism underlining the inhibition of eEF2K/eEF2 pathway in rescuing epileptic and behavioral alterations in the Scn1a ± needs further investigations. CONCLUSIONS: Our data indicate that pharmacological inhibition of eEF2K could represent a novel therapeutic intervention for treating epilepsy and related comorbidities in the Dravet syndrome.

Loss of ARHGAP15 affects the directional control of migrating interneurons in the embryonic cortex and increases susceptibility to epilepsy.

GTPases of the Rho family are components of signaling pathways linking extracellular signals to the control of cytoskeleton dynamics. Among these, RAC1 plays key roles during brain development, ranging from neuronal migration to neuritogenesis, synaptogenesis, and plasticity. RAC1 activity is positively and negatively controlled by guanine nucleotide exchange factors (GEFs), guanosine nucleotide dissociation inhibitors (GDIs), and GTPase-activating proteins (GAPs), but the specific role of each regulator in vivo is poorly known. ARHGAP15 is a RAC1-specific GAP expressed during development in a fraction of migrating cortical interneurons (CINs) and in the majority of adult CINs. During development, loss of ARHGAP15 causes altered directionality of the leading process of tangentially migrating CINs, along with altered morphology in vitro. Likewise, time-lapse imaging of embryonic CINs revealed a poorly coordinated directional control during radial migration, possibly due to a hyper-exploratory behavior. In the adult cortex, the observed defects lead to subtle alteration in the distribution of CALB2-, SST-, and VIP-positive interneurons. Adult Arhgap15-knock-out mice also show reduced CINs intrinsic excitability, spontaneous subclinical seizures, and increased susceptibility to the pro-epileptic drug pilocarpine. These results indicate that ARHGAP15 imposes a fine negative regulation on RAC1 that is required for morphological maturation and directional control during CIN migration, with consequences on their laminar distribution and inhibitory function.

Expression of mutant beta2 nicotinic receptors during development is crucial for epileptogenesis.

Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is a focal form of epilepsy characterized by seizures occurring during non-REM sleep. We have developed and characterized the first mouse model for ADNFLE type III carrying the V287L mutation of the beta2 subunit of neuronal nicotinic receptor. Mice expressing mutant receptors show a spontaneous epileptic phenotype by electroencephalography with very frequent interictal spikes and seizures. Expression of the mutant beta2 subunit is driven by a neuronal-specific tetracycline-controlled promoter, which allows planned silencing of transgene expression in a reversible fashion and tracking the involvement of mutant receptor in crucial phases of epileptogenesis. We found that restricted silencing during development is sufficient to prevent the occurrence of epileptic seizures in adulthood. Our data indicate that mutant nicotinic receptors are responsible for abnormal formation of neuronal circuits and/or long-lasting alteration of network assembly in the developing brain, thus leading to epilepsy.