Anti-seizure gene therapy for focal cortical dysplasia.

Focal cortical dysplasias are a common subtype of malformation of cortical development, which frequently presents with a spectrum of cognitive and behavioural abnormalities as well as pharmacoresistant epilepsy. Focal cortical dysplasia type II is typically caused by somatic mutations resulting in mammalian target of rapamycin (mTOR) hyperactivity, and is the commonest pathology found in children undergoing epilepsy surgery. However, surgical resection does not always result in seizure freedom, and is often precluded by proximity to eloquent brain regions. Gene therapy is a promising potential alternative treatment and may be appropriate in cases that represent an unacceptable surgical risk. Here, we evaluated a gene therapy based on overexpression of the Kv1.1 potassium channel in a mouse model of frontal lobe focal cortical dysplasia. An engineered potassium channel (EKC) transgene was placed under control of a human promoter that biases expression towards principal neurons (CAMK2A) and packaged in an adeno-associated viral vector (AAV9). We used an established focal cortical dysplasia model generated by in utero electroporation of frontal lobe neural progenitors with a constitutively active human Ras homolog enriched in brain (RHEB) plasmid, an activator of mTOR complex 1. We characterized the model by quantifying electrocorticographic and behavioural abnormalities, both in mice developing spontaneous generalized seizures and in mice only exhibiting interictal discharges. Injection of AAV9-CAMK2A-EKC in the dysplastic region resulted in a robust decrease (∼64%) in the frequency of seizures. Despite the robust anti-epileptic effect of the treatment, there was neither an improvement nor a worsening of performance in behavioural tests sensitive to frontal lobe function. AAV9-CAMK2A-EKC had no effect on interictal discharges or behaviour in mice without generalized seizures. AAV9-CAMK2A-EKC gene therapy is a promising therapy with translational potential to treat the epileptic phenotype of mTOR-related malformations of cortical development. Cognitive and behavioural co-morbidities may, however, resist an intervention aimed at reducing circuit excitability.

Neurodevelopmental and synaptic defects in DNAJC6 parkinsonism, amenable to gene therapy.

DNAJC6 encodes auxilin, a co-chaperone protein involved in clathrin-mediated endocytosis (CME) at the presynaptic terminal. Biallelic mutations in DNAJC6 cause a complex, early-onset neurodegenerative disorder characterized by rapidly progressive parkinsonism-dystonia in childhood. The disease is commonly associated with additional neurodevelopmental, neurological and neuropsychiatric features. Currently, there are no disease-modifying treatments for this condition, resulting in significant morbidity and risk of premature mortality. To investigate the underlying disease mechanisms in childhood-onset DNAJC6 parkinsonism, we generated induced pluripotent stem cells (iPSC) from three patients harbouring pathogenic loss-of-function DNAJC6 mutations and subsequently developed a midbrain dopaminergic neuronal model of disease. When compared to age-matched and CRISPR-corrected isogenic controls, the neuronal cell model revealed disease-specific auxilin deficiency as well as disturbance of synaptic vesicle recycling and homeostasis. We also observed neurodevelopmental dysregulation affecting ventral midbrain patterning and neuronal maturation. To explore the feasibility of a viral vector-mediated gene therapy approach, iPSC-derived neuronal cultures were treated with lentiviral DNAJC6 gene transfer, which restored auxilin expression and rescued CME. Our patient-derived neuronal model provides deeper insights into the molecular mechanisms of auxilin deficiency as well as a robust platform for the development of targeted precision therapy approaches.

Ca2+ binding to synapsin I regulates resting Ca2+ and recovery from synaptic depression in nerve terminals.

Synapsin I (SynI) is a synaptic vesicle (SV)-associated phosphoprotein that modulates neurotransmission by controlling SV trafficking. The SynI C-domain contains a highly conserved ATP binding site mediating SynI oligomerization and SV clustering and an adjacent main Ca2+ binding site, whose physiological role is unexplored. Molecular dynamics simulations revealed that the E373K point mutation irreversibly deletes Ca2+ binding to SynI, still allowing ATP binding, but inducing a destabilization of the SynI oligomerization interface. Here, we analyzed the effects of this mutation on neurotransmitter release and short-term plasticity in excitatory and inhibitory synapses from primary hippocampal neurons. Patch-clamp recordings showed an increase in the frequency of miniature excitatory postsynaptic currents (EPSCs) that was totally occluded by exogenous Ca2+ chelators and associated with a constitutive increase in resting terminal Ca2+ concentrations. Evoked EPSC amplitude was also reduced, due to a decreased readily releasable pool (RRP) size. Moreover, in both excitatory and inhibitory synapses, we observed a marked impaired recovery from synaptic depression, associated with impaired RRP refilling and depletion of the recycling pool of SVs. Our study identifies SynI as a novel Ca2+ buffer in excitatory terminals. Blocking Ca2+ binding to SynI results in higher constitutive Ca2+ levels that increase the probability of spontaneous release and disperse SVs. This causes a decreased size of the RRP and an impaired recovery from depression due to the failure of SV reclustering after sustained high-frequency stimulation. The results indicate a physiological role of Ca2+ binding to SynI in the regulation of SV clustering and trafficking in nerve terminals.

Aromatic l-amino acid decarboxylase deficiency: a patient-derived neuronal model for precision therapies.

Aromatic l-amino acid decarboxylase (AADC) deficiency is a complex inherited neurological disorder of monoamine synthesis which results in dopamine and serotonin deficiency. The majority of affected individuals have variable, though often severe cognitive and motor delay, with a complex movement disorder and high risk of premature mortality. For most, standard pharmacological treatment provides only limited clinical benefit. Promising gene therapy approaches are emerging, though may not be either suitable or easily accessible for all patients. To characterize the underlying disease pathophysiology and guide precision therapies, we generated a patient-derived midbrain dopaminergic neuronal model of AADC deficiency from induced pluripotent stem cells. The neuronal model recapitulates key disease features, including absent AADC enzyme activity and dysregulated dopamine metabolism. We observed developmental defects affecting synaptic maturation and neuronal electrical properties, which were improved by lentiviral gene therapy. Bioinformatic and biochemical analyses on recombinant AADC predicted that the activity of one variant could be improved by l-3,4-dihydroxyphenylalanine (l-DOPA) administration; this hypothesis was corroborated in the patient-derived neuronal model, where l-DOPA treatment leads to amelioration of dopamine metabolites. Our study has shown that patient-derived disease modelling provides further insight into the neurodevelopmental sequelae of AADC deficiency, as well as a robust platform to investigate and develop personalized therapeutic approaches.

Recent advances in gene therapy for neurodevelopmental disorders with epilepsy.

Neurodevelopmental disorders can be caused by mutations in neuronal genes fundamental to brain development. These disorders have severe symptoms ranging from intellectually disability, social and cognitive impairments, and a subset are strongly linked with epilepsy. In this review, we focus on those neurodevelopmental disorders that are frequently characterized by the presence of epilepsy (NDD + E). We loosely group the genes linked to NDD + E with different neuronal functions: transcriptional regulation, intrinsic excitability and synaptic transmission. All these genes have in common a pivotal role in defining the brain architecture and function during early development, and when their function is altered, symptoms can present in the first stages of human life. The relationship with epilepsy is complex. In some NDD + E, epilepsy is a comorbidity and in others seizures appear to be the main cause of the pathology, suggesting that either structural changes (NDD) or neuronal communication (E) can lead to these disorders. Furthermore, grouping the genes that cause NDD + E, we review the uses and limitations of current models of the different disorders, and how different gene therapy strategies are being developed to treat them. We highlight where gene replacement may not be a treatment option, and where innovative therapeutic tools, such as CRISPR-based gene editing, and new avenues of delivery are required. In general this group of genetically defined disorders, supported increasing knowledge of the mechanisms leading to neurological dysfunction serve as an excellent collection for illustrating the translational potential of gene therapy, including newly emerging tools.

Progressive myoclonus epilepsy KCNC1 variant causes a developmental dendritopathy.

OBJECTIVE: Mutations in KCNC1 can cause severe neurological dysfunction, including intellectual disability, epilepsy, and ataxia. The Arg320His variant, which occurs in the voltage-sensing domain of the channel, causes a highly penetrant and specific form of progressive myoclonus epilepsy with severe ataxia, designated myoclonus epilepsy and ataxia due to potassium channel mutation (MEAK). KCNC1 encodes the voltage-gated potassium channel KV 3.1, a channel that is important for enabling high-frequency firing in interneurons, raising the possibility that MEAK is associated with reduced interneuronal function. METHODS: To determine how this variant triggers MEAK, we expressed KV 3.1bR320H in cortical interneurons in vitro and investigated the effects on neuronal function and morphology. We also performed electrophysiological recordings of oocytes expressing KV 3.1b to determine whether the mutation introduces gating pore currents. RESULTS: Expression of the KV 3.1bR320H variant profoundly reduced excitability of mature cortical interneurons, and cells expressing these channels were unable to support high-frequency firing. The mutant channel also had an unexpected effect on morphology, severely impairing neurite development and interneuron viability, an effect that could not be rescued by blocking KV 3 channels. Oocyte recordings confirmed that in the adult KV 3.1b isoform, R320H confers a dominant negative loss-of-function effect by slowing channel activation, but does not introduce potentially toxic gating pore currents. SIGNIFICANCE: Overall, our data suggest that, in addition to the regulation of high-frequency firing, KV 3.1 channels play a hitherto unrecognized role in neuronal development. MEAK may be described as a developmental dendritopathy.

In vivo CRISPRa decreases seizures and rescues cognitive deficits in a rodent model of epilepsy.

Epilepsy is a major health burden, calling for new mechanistic insights and therapies. CRISPR-mediated gene editing shows promise to cure genetic pathologies, although hitherto it has mostly been applied ex vivo. Its translational potential for treating non-genetic pathologies is still unexplored. Furthermore, neurological diseases represent an important challenge for the application of CRISPR, because of the need in many cases to manipulate gene function of neurons in situ. A variant of CRISPR, CRISPRa, offers the possibility to modulate the expression of endogenous genes by directly targeting their promoters. We asked if this strategy can effectively treat acquired focal epilepsy, focusing on ion channels because their manipulation is known be effective in changing network hyperactivity and hypersynchronziation. We applied a doxycycline-inducible CRISPRa technology to increase the expression of the potassium channel gene Kcna1 (encoding Kv1.1) in mouse hippocampal excitatory neurons. CRISPRa-mediated Kv1.1 upregulation led to a substantial decrease in neuronal excitability. Continuous video-EEG telemetry showed that AAV9-mediated delivery of CRISPRa, upon doxycycline administration, decreased spontaneous generalized tonic-clonic seizures in a model of temporal lobe epilepsy, and rescued cognitive impairment and transcriptomic alterations associated with chronic epilepsy. The focal treatment minimizes concerns about off-target effects in other organs and brain areas. This study provides the proof-of-principle for a translational CRISPR-based approach to treat neurological diseases characterized by abnormal circuit excitability.

dCas9-Based Scn1a Gene Activation Restores Inhibitory Interneuron Excitability and Attenuates Seizures in Dravet Syndrome Mice.

Dravet syndrome (DS) is a severe epileptic encephalopathy caused mainly by heterozygous loss-of-function mutations of the SCN1A gene, indicating haploinsufficiency as the pathogenic mechanism. Here we tested whether catalytically dead Cas9 (dCas9)-mediated Scn1a gene activation can rescue Scn1a haploinsufficiency in a mouse DS model and restore physiological levels of its gene product, the Nav1.1 voltage-gated sodium channel. We screened single guide RNAs (sgRNAs) for their ability to stimulate Scn1a transcription in association with the dCas9 activation system. We identified a specific sgRNA that increases Scn1a gene expression levels in cell lines and primary neurons with high specificity. Nav1.1 protein levels were augmented, as was the ability of wild-type immature GABAergic interneurons to fire action potentials. A similar enhancement of Scn1a transcription was achieved in mature DS interneurons, rescuing their ability to fire. To test the therapeutic potential of this approach, we delivered the Scn1a-dCas9 activation system to DS pups using adeno-associated viruses. Parvalbumin interneurons recovered their firing ability, and febrile seizures were significantly attenuated. Our results pave the way for exploiting dCas9-based gene activation as an effective and targeted approach to DS and other disorders resulting from altered gene dosage.

LGI1 downregulation increases neuronal circuit excitability.

OBJECTIVE: Leucine-rich glioma-inactivated 1 (LGI1) is a secreted transsynaptic protein that interacts presynaptically with Kv1.1 potassium channels and a disintegrin and metalloprotease (ADAM) protein 23, and postsynaptically influences α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors through a direct link with the ADAM22 cell adhesion protein. Haploinsufficiency of LGI1 or autoantibodies directed against LGI1 are associated with human epilepsy, generating the hypothesis that a subacute reduction of LGI1 is sufficient to increase network excitability. METHODS: We tested this hypothesis in ex vivo hippocampal slices and in neuronal cultures, by subacutely reducing LGI1 expression with shRNA. RESULTS: Injection of shRNA-LGI1 in the hippocampus increased dentate granule cell excitability and low-frequency facilitation of mossy fibers to CA3 pyramidal cell neurotransmission. Application of the Kv1 family blocker, α-dendrotoxin, occluded this effect, implicating the involvement of Kv1.1. This subacute reduction of LGI1 was also sufficient to increase neuronal network activity in neuronal primary culture. SIGNIFICANCE: These results indicate that a subacute reduction in LGI1 potentiates neuronal excitability and short-term synaptic plasticity, and increases neuronal network excitability, opening new avenues for the treatment of limbic encephalitis and temporal lobe epilepsies.

Activity Clamp Provides Insights into Paradoxical Effects of the Anti-Seizure Drug Carbamazepine.

A major challenge in experimental epilepsy research is to reconcile the effects of anti-epileptic drugs (AEDs) on individual neurons with their network-level actions. Highlighting this difficulty, it is unclear why carbamazepine (CBZ), a frontline AED with a known molecular mechanism, has been reported to increase epileptiform activity in several clinical and experimental studies. We confirmed in an in vitro mouse model (in both sexes) that the frequency of interictal bursts increased after CBZ perfusion. To address the underlying mechanisms, we developed a method, activity clamp, to distinguish the response of individual neurons from network-level actions of CBZ. We first recorded barrages of synaptic conductances from neurons during epileptiform activity and then replayed them in pharmacologically isolated neurons under control conditions and in the presence of CBZ. CBZ consistently decreased the reliability of the second action potential in each burst of activity. Conventional current-clamp recordings using excitatory ramp or square-step current injections failed to reveal this effect. Network modeling showed that a CBZ-induced decrease of neuron recruitment during epileptic bursts can lead to an increase in burst frequency at the network level by reducing the refractoriness of excitatory transmission. By combining activity clamp with computer simulations, the present study provides a potential explanation for the paradoxical effects of CBZ on epileptiform activity.SIGNIFICANCE STATEMENT The effects of anti-epileptic drugs on individual neurons are difficult to separate from their network-level actions. Although carbamazepine (CBZ) has a known anti-epileptic mechanism, paradoxically, it has also been reported to increase epileptiform activity in clinical and experimental studies. To investigate this paradox during realistic neuronal epileptiform activity, we developed a method, activity clamp, to distinguish the effects of CBZ on individual neurons from network-level actions. We demonstrate that CBZ consistently decreases the reliability of the second action potential in each burst of epileptiform activity. Network modeling shows that this effect on individual neuronal responses could explain the paradoxical effect of CBZ at the network level.

Cell adhesion molecule L1 contributes to neuronal excitability regulating the function of voltage-gated Na+ channels.

L1 (also known as L1CAM) is a trans-membrane glycoprotein mediating neuron-neuron adhesion through homophilic and heterophilic interactions. Although experimental evidence has implicated L1 in axonal outgrowth, fasciculation and pathfinding, its contribution to voltage-gated Na(+) channel function and membrane excitability has remained unknown. Here, we show that firing rate, single cell spiking frequency and Na(+) current density are all reduced in hippocampal excitatory neurons from L1-deficient mice both in culture and in slices owing to an overall reduced membrane expression of Na(+) channels. Remarkably, normal firing activity was restored when L1 was reintroduced into L1-deficient excitatory neurons, indicating that abnormal firing patterns are not related to developmental abnormalities, but are a direct consequence of L1 deletion. Moreover, L1 deficiency leads to impairment of action potential initiation, most likely due to the loss of the interaction of L1 with ankyrin G that produces the delocalization of Na(+) channels at the axonal initial segment. We conclude that L1 contributes to functional expression and localization of Na(+) channels to the neuronal plasma membrane, ensuring correct initiation of action potential and normal firing activity.

REST/NRSF-mediated intrinsic homeostasis protects neuronal networks from hyperexcitability.

Intrinsic homeostasis enables neuronal circuits to maintain activity levels within an appropriate range by modulating neuronal voltage-gated conductances, but the signalling pathways involved in this process are largely unknown. We characterized the process of intrinsic homeostasis induced by sustained electrical activity in cultured hippocampal neurons based on the activation of the Repressor Element-1 Silencing Transcription Factor/Neuron-Restrictive Silencer Factor (REST/NRSF). We showed that 4-aminopyridine-induced hyperactivity enhances the expression of REST/NRSF, which in turn, reduces the expression of voltage-gated Na(+) channels, thereby decreasing the neuronal Na(+) current density. This mechanism plays an important role in the downregulation of the firing activity at the single-cell level, re-establishing a physiological spiking activity in the entire neuronal network. Conversely, interfering with REST/NRSF expression impaired this homeostatic response. Our results identify REST/NRSF as a critical factor linking neuronal activity to the activation of intrinsic homeostasis and restoring a physiological level of activity in the entire neuronal network.

Functional role of ATP binding to synapsin I in synaptic vesicle trafficking and release dynamics.

Synapsins (Syns) are synaptic vesicle (SV)-associated proteins involved in the regulation of synaptic transmission and plasticity, which display a highly conserved ATP binding site in the central C-domain, whose functional role is unknown. Using molecular dynamics simulations, we demonstrated that ATP binding to SynI is mediated by a conformational transition of a flexible loop that opens to make the binding site accessible; such transition, prevented in the K269Q mutant, is not significantly affected in the absence of Ca(2+) or by the E373K mutation that abolishes Ca(2+)-binding. Indeed, the ATP binding to SynI also occurred under Ca(2+)-free conditions and increased its association with purified rat SVs regardless of the presence of Ca(2+) and promoted SynI oligomerization. However, although under Ca(2+)-free conditions, SynI dimerization and SV clustering were enhanced, Ca(2+) favored the formation of tetramers at the expense of dimers and did not affect SV clustering, indicating a role of Ca(2+)-dependent dimer/tetramer transitions in the regulation of ATP-dependent SV clustering. To elucidate the role of ATP/SynI binding in synaptic physiology, mouse SynI knock-out hippocampal neurons were transduced with either wild-type or K269Q mutant SynI and inhibitory transmission was studied by patch-clamp and electron microscopy. K269Q-SynI expressing inhibitory synapses showed increased synaptic strength due to an increase in the release probability, an increased vulnerability to synaptic depression and a dysregulation of SV trafficking, when compared with wild-type SynI-expressing terminals. The results suggest that the ATP-SynI binding plays predocking and postdocking roles in the modulation of SV clustering and plasticity of inhibitory synapses.

Phosphorylation of synapsin I by cyclin-dependent kinase-5 sets the ratio between the resting and recycling pools of synaptic vesicles at hippocampal synapses.

Cyclin-dependent kinase-5 (Cdk5) was reported to downscale neurotransmission by sequestering synaptic vesicles (SVs) in the release-reluctant resting pool, but the molecular targets mediating this activity remain unknown. Synapsin I (SynI), a major SV phosphoprotein involved in the regulation of SV trafficking and neurotransmitter release, is one of the presynaptic substrates of Cdk5, which phosphorylates it in its C-terminal region at Ser(549) (site 6) and Ser(551) (site 7). Here we demonstrate that Cdk5 phosphorylation of SynI fine tunes the recruitment of SVs to the active recycling pool and contributes to the Cdk5-mediated homeostatic responses. Phosphorylation of SynI by Cdk5 is physiologically regulated and enhances its binding to F-actin. The effects of Cdk5 inhibition on the size and depletion kinetics of the recycling pool, as well as on SV distribution within the nerve terminal, are virtually abolished in mouse SynI knock-out (KO) neurons or in KO neurons expressing the dephosphomimetic SynI mutants at sites 6,7 or site 7 only. The observation that the single site-7 mutant phenocopies the effects of the deletion of SynI identifies this site as the central switch in mediating the synaptic effects of Cdk5 and demonstrates that SynI is necessary and sufficient for achieving the effects of the kinase on SV trafficking. The phosphorylation state of SynI by Cdk5 at site 7 is regulated during chronic modification of neuronal activity and is an essential downstream effector for the Cdk5-mediated homeostatic scaling.

Epileptogenic Q555X SYN1 mutant triggers imbalances in release dynamics and short-term plasticity.

Synapsin I (SynI) is a synaptic vesicle (SV) phosphoprotein playing multiple roles in synaptic transmission and plasticity by differentially affecting crucial steps of SV trafficking in excitatory and inhibitory synapses. SynI knockout (KO) mice are epileptic, and nonsense and missense mutations in the human SYN1 gene have a causal role in idiopathic epilepsy and autism. To get insights into the mechanisms of epileptogenesis linked to SYN1 mutations, we analyzed the effects of the recently identified Q555X mutation on neurotransmitter release dynamics and short-term plasticity (STP) in excitatory and inhibitory synapses. We used patch-clamp electrophysiology coupled to electron microscopy and multi-electrode arrays to dissect synaptic transmission of primary SynI KO hippocampal neurons in which the human wild-type and mutant SynI were expressed by lentiviral transduction. A parallel decrease in the SV readily releasable pool in inhibitory synapses and in the release probability in excitatory synapses caused a marked reduction in the evoked synchronous release. This effect was accompanied by an increase in asynchronous release that was much more intense in excitatory synapses and associated with an increased total charge transfer. Q555X-hSynI induced larger facilitation and post-tetanic potentiation in excitatory synapses and stronger depression after long trains in inhibitory synapses. These changes were associated with higher network excitability and firing/bursting activity. Our data indicate that imbalances in STP and release dynamics of inhibitory and excitatory synapses trigger network hyperexcitability potentially leading to epilepsy/autism manifestations.

Synapsins: from synapse to network hyperexcitability and epilepsy.

The synapsin family in mammals consists of at least 10 isoforms encoded by three distinct genes and composed by a mosaic of conserved and variable domains. Synapsins, although not essential for the basic development and functioning of neuronal networks, are extremely important for the fine-tuning of SV cycling and neuronal plasticity. Single, double and triple synapsin knockout mice, with the notable exception of the synapsin III knockout mice, show a severe epileptic phenotype without gross alterations in brain morphology and connectivity. However, the molecular and physiological mechanisms underlying the pathogenesis of the epileptic phenotype observed in synapsin deficient mice are still far from being elucidated. In this review, we summarize the current knowledge about the role of synapsins in the regulation of network excitability and about the molecular mechanism leading to epileptic phenotype in mouse lines lacking one or more synapsin isoforms. The current evidences indicate that synapsins exert distinct roles in excitatory versus inhibitory synapses by differentially affecting crucial steps of presynaptic physiology and by this mean participate in the determination of network hyperexcitability.

Are Genetic Therapies for Epilepsy Ready for the Clinic?

In recent years, there has been a significant increase in preclinical studies to test genetic therapies for epilepsy. Some of these therapies have advanced to clinical trials and are being tested in patients with monogenetic or focal refractory epilepsy. This article provides an overview of the current state of preclinical studies that show potential for clinical translation. Specifically, we focus on genetic therapies that have demonstrated a clear effect on seizures in animal models and have the potential to be translated to clinical settings. Both therapies targeting the cause of the disease and those that treat symptoms are discussed. We believe that the next few years will be crucial in determining the potential of genetic therapies for treating patients with epilepsy.

Epilepsy-linked kinase CDKL5 phosphorylates voltage-gated calcium channel Cav2.3, altering inactivation kinetics and neuronal excitability.

Developmental and epileptic encephalopathies (DEEs) are a group of rare childhood disorders characterized by severe epilepsy and cognitive deficits. Numerous DEE genes have been discovered thanks to advances in genomic diagnosis, yet putative molecular links between these disorders are unknown. CDKL5 deficiency disorder (CDD, DEE2), one of the most common genetic epilepsies, is caused by loss-of-function mutations in the brain-enriched kinase CDKL5. To elucidate CDKL5 function, we looked for CDKL5 substrates using a SILAC-based phosphoproteomic screen. We identified the voltage-gated Ca2+ channel Cav2.3 (encoded by CACNA1E) as a physiological target of CDKL5 in mice and humans. Recombinant channel electrophysiology and interdisciplinary characterization of Cav2.3 phosphomutant mice revealed that loss of Cav2.3 phosphorylation leads to channel gain-of-function via slower inactivation and enhanced cholinergic stimulation, resulting in increased neuronal excitability. Our results thus show that CDD is partly a channelopathy. The properties of unphosphorylated Cav2.3 closely resemble those described for CACNA1E gain-of-function mutations causing DEE69, a disorder sharing clinical features with CDD. We show that these two single-gene diseases are mechanistically related and could be ameliorated with Cav2.3 inhibitors.

Strategies to maximize the performance of a STED microscope.

In stimulated emission depletion (STED) microscopy, the spatial resolution scales as the inverse square root of the STED beam's intensity. However, to fully exploit the maximum effective resolution achievable for a given STED beam's intensity, several experimental precautions have to be considered. We focus our attention on the temporal alignment between the excitation and STED pulses and the polarization state of the STED beam. We present a simple theoretical framework that help to explain their influence on the performance of a STED microscope and we validate the results by imaging calibration and biological samples with a custom made STED architecture based on a supercontinuum laser source. We also highlight the advantages of using time gating detection in terms of temporal alignment.