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.

A branching model of lineage differentiation underpinning the neurogenic potential of enteric glia.

Glial cells have been proposed as a source of neural progenitors, but the mechanisms underpinning the neurogenic potential of adult glia are not known. Using single cell transcriptomic profiling, we show that enteric glial cells represent a cell state attained by autonomic neural crest cells as they transition along a linear differentiation trajectory that allows them to retain neurogenic potential while acquiring mature glial functions. Key neurogenic loci in early enteric nervous system progenitors remain in open chromatin configuration in mature enteric glia, thus facilitating neuronal differentiation under appropriate conditions. Molecular profiling and gene targeting of enteric glial cells in a cell culture model of enteric neurogenesis and a gut injury model demonstrate that neuronal differentiation of glia is driven by transcriptional programs employed in vivo by early progenitors. Our work provides mechanistic insight into the regulatory landscape underpinning the development of intestinal neural circuits and generates a platform for advancing glial cells as therapeutic agents for the treatment of neural deficits.

Activity-dependent membrane sculpting deficits in TAOK1-linked neurodevelopmental disease.

A recent study by Beeman et al. exploring disease-related missense mutations in TAOK1 revealed a self-regulating association of the kinase with the plasma membrane that is critical for neuronal morphogenesis. Using a combination of in vitro approaches and elegant in silico modeling, the authors describe an aberrant membrane protrusions phenotype in kinase-deficient mutants reminiscent of TAOK2's indirect regulation of neuronal morphology, thus providing a converging patho-mechanism across several neurodevelopmental disorders.

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.

Myotonia in a patient with a mutation in an S4 arginine residue associated with hypokalaemic periodic paralysis and a concomitant synonymous CLCN1 mutation.

The sarcolemmal voltage gated sodium channel NaV1.4 conducts the key depolarizing current that drives the upstroke of the skeletal muscle action potential. It contains four voltage-sensing domains (VSDs) that regulate the opening of the pore domain and ensuing permeation of sodium ions. Mutations that lead to increased NaV1.4 currents are found in patients with myotonia or hyperkalaemic periodic paralysis (HyperPP). Myotonia is also caused by mutations in the CLCN1gene that result in loss-of-function of the skeletal muscle chloride channel ClC-1. Mutations affecting arginine residues in the fourth transmembrane helix (S4) of the NaV1.4 VSDs can result in a leak current through the VSD and hypokalemic periodic paralysis (HypoPP), but these have hitherto not been associated with myotonia. We report a patient with an Nav1.4 S4 arginine mutation, R222Q, presenting with severe myotonia without fulminant paralytic episodes. Other mutations affecting the same residue, R222W and R222G, have been found in patients with HypoPP. We show that R222Q channels have enhanced activation, consistent with myotonia, but also conduct a leak current. The patient carries a concomitant synonymous CLCN1 variant that likely worsens the myotonia and potentially contributes to the amelioration of muscle paralysis. Our data show phenotypic variability for different mutations affecting the same S4 arginine that have implications for clinical therapy.

Benzamil inhibits neuronal and heterologously expressed small conductance Ca2+-activated K+ channels.

Small conductance Ca2+-activated K+ (SK) channels are expressed throughout the soma and dendrites of pyramidal neurons in the neocortex and hippocampal formation, where they participate in the local regulation of membrane excitability and synaptic signals. Through their inter-play with Ca2+ channels, SK channels regulate Ca2+ influx triggered by back-propagating action potentials in dendrites. Inhibition of SK channels affects both the amplitude and duration of Ca2+ transients, but the role of Ca2+ clearance mechanisms and their link to SK channel activity has not been established. Here we report the effect of the Na+/Ca2+ exchanger (NCX) inhibitor benzamil on Ca2+ extrusion and SK channels in the regulation of dendritic Ca2+ signals. Benzamil increased the duration and amplitude of dendritic Ca2+ transients elicited by back-propagating action potentials in hippocampal pyramidal neurons. This data is consistent with previous studies with SK channel blockers and suggests that benzamil inhibits SK channels in addition to the Na+/Ca2+ exchanger. Here we show that indeed both the neuronal SK-mediated IAHP current and the currents mediated by heterologously expressed SK channels were inhibited by benzamil. The inhibition of recombinant SK channels was seen with different K+ concentration gradients, and was stronger at negative voltages. The suppression of SK channels by benzamil is consistent with previous findings on the modulation of Ca2+ signals by SK channels in neurons. We additionally show that benzamil inhibits neuronal voltage-gated calcium currents. The results prompt a careful reassessment of the effects of benzamil on Ca2+ transients in native systems, given the spectrum of ion channels and exchangers this compound targets within a similar range of concentrations.

Myasthenic congenital myopathy from recessive mutations at a single residue in NaV1.4.

OBJECTIVE: To identify the genetic and physiologic basis for recessive myasthenic congenital myopathy in 2 families, suggestive of a channelopathy involving the sodium channel gene, SCN4A. METHODS: A combination of whole exome sequencing and targeted mutation analysis, followed by voltage-clamp studies of mutant sodium channels expressed in fibroblasts (HEK cells) and Xenopus oocytes. RESULTS: Missense mutations of the same residue in the skeletal muscle sodium channel, R1460 of NaV1.4, were identified in a family and a single patient of Finnish origin (p.R1460Q) and a proband in the United States (p.R1460W). Congenital hypotonia, breathing difficulties, bulbar weakness, and fatigability had recessive inheritance (homozygous p.R1460W or compound heterozygous p.R1460Q and p.R1059X), whereas carriers were either asymptomatic (p.R1460W) or had myotonia (p.R1460Q). Sodium currents conducted by mutant channels showed unusual mixed defects with both loss-of-function (reduced amplitude, hyperpolarized shift of inactivation) and gain-of-function (slower entry and faster recovery from inactivation) changes. CONCLUSIONS: Novel mutations in families with myasthenic congenital myopathy have been identified at p.R1460 of the sodium channel. Recessive inheritance, with experimentally established loss-of-function, is a consistent feature of sodium channel based myasthenia, whereas the mixed gain of function for p.R1460 may also cause susceptibility to myotonia.

A novel ATP1A2 mutation in a patient with hypokalaemic periodic paralysis and CNS symptoms.

Hypokalaemic periodic paralysis is a rare genetic neuromuscular disease characterized by episodes of skeletal muscle paralysis associated with low serum potassium. Muscle fibre inexcitability during attacks of paralysis is due to an aberrant depolarizing leak current through mutant voltage sensing domains of either the sarcolemmal voltage-gated calcium or sodium channel. We report a child with hypokalaemic periodic paralysis and CNS involvement, including seizures, but without mutations in the known periodic paralysis genes. We identified a novel heterozygous de novo missense mutation in the ATP1A2 gene encoding the α2 subunit of the Na+/K+-ATPase that is abundantly expressed in skeletal muscle and in brain astrocytes. Pump activity is crucial for Na+ and K+ homeostasis following sustained muscle or neuronal activity and its dysfunction is linked to the CNS disorders hemiplegic migraine and alternating hemiplegia of childhood, but muscle dysfunction has not been reported. Electrophysiological measurements of mutant pump activity in Xenopus oocytes revealed lower turnover rates in physiological extracellular K+ and an anomalous inward leak current in hypokalaemic conditions, predicted to lead to muscle depolarization. Our data provide important evidence supporting a leak current as the major pathomechanism underlying hypokalaemic periodic paralysis and indicate ATP1A2 as a new hypokalaemic periodic paralysis gene.

Hypokalaemic periodic paralysis and myotonia in a patient with homozygous mutation p.R1451L in NaV1.4.

Dominantly inherited channelopathies of the skeletal muscle voltage-gated sodium channel NaV1.4 include hypokalaemic and hyperkalaemic periodic paralysis (hypoPP and hyperPP) and myotonia. HyperPP and myotonia are caused by NaV1.4 channel overactivity and overlap clinically. Instead, hypoPP is caused by gating pore currents through the voltage sensing domains (VSDs) of NaV1.4 and seldom co-exists clinically with myotonia. Recessive loss-of-function NaV1.4 mutations have been described in congenital myopathy and myasthenic syndromes. We report two families with the NaV1.4 mutation p.R1451L, located in VSD-IV. Heterozygous carriers in both families manifest with myotonia and/or hyperPP. In contrast, a homozygous case presents with both hypoPP and myotonia, but unlike carriers of recessive NaV1.4 mutations does not manifest symptoms of myopathy or myasthenia. Functional analysis revealed reduced current density and enhanced closed state inactivation of the mutant channel, but no evidence for gating pore currents. The rate of recovery from inactivation was hastened, explaining the myotonia in p.R1451L carriers and the absence of myasthenic presentations in the homozygous proband. Our data suggest that recessive loss-of-function NaV1.4 variants can present with hypoPP without congenital myopathy or myasthenia and that myotonia can present even in carriers of homozygous NaV1.4 loss-of-function mutations.

Spider toxin inhibits gating pore currents underlying periodic paralysis.

Gating pore currents through the voltage-sensing domains (VSDs) of the skeletal muscle voltage-gated sodium channel NaV1.4 underlie hypokalemic periodic paralysis (HypoPP) type 2. Gating modifier toxins target ion channels by modifying the function of the VSDs. We tested the hypothesis that these toxins could function as blockers of the pathogenic gating pore currents. We report that a crab spider toxin Hm-3 from Heriaeus melloteei can inhibit gating pore currents due to mutations affecting the second arginine residue in the S4 helix of VSD-I that we have found in patients with HypoPP and describe here. NMR studies show that Hm-3 partitions into micelles through a hydrophobic cluster formed by aromatic residues and reveal complex formation with VSD-I through electrostatic and hydrophobic interactions with the S3b helix and the S3-S4 extracellular loop. Our data identify VSD-I as a specific binding site for neurotoxins on sodium channels. Gating modifier toxins may constitute useful hits for the treatment of HypoPP.

In situ monitoring of PTHLH secretion in neuroblastoma cells cultured onto nanoporous membranes.

In this work, we propose for the first time the use of anodic aluminum oxide (AAO) nanoporous membranes for in situ monitoring of parathyroid hormone-like hormone (PTHLH) secretion in cultured human cells. The biosensing system is based on the nanochannels blockage upon immunocomplex formation, which is electrically monitored through the voltammetric oxidation of Prussian blue nanoparticles (PBNPs). Models evaluated include a neuroblastoma cell line (SK-N-AS) and immortalized keratinocytes (HaCaT) as a control of high PTHLH production. The effect of total number of seeded cells and incubation time on the secreted PTHLH levels is assessed, finding that secreted PTHLH levels range from approximately 60 to 400 ng/mL. Moreover, our methodology is also applied to analyse PTHLH production following PTHLH gene knockdown upon transient cell transfection with a specific silencing RNA (siRNA). Given that inhibition of PTHLH secretion reduces cell proliferation, survival and invasiveness in a number of tumors, our system provides a powerful tool for the preclinical evaluation of therapies that regulate PTHLH production. This nanoporous membrane - based sensing technology might be useful to monitor the active secretion of other proteins as well, thus contributing to characterize their regulation and function.

Chemical genetic identification of GAK substrates reveals its role in regulating Na+/K+-ATPase.

Cyclin G-associated kinase (GAK) is a ubiquitous serine/threonine kinase that facilitates clathrin uncoating during vesicle trafficking. GAK phosphorylates a coat adaptor component, AP2M1, to help achieve this function. GAK is also implicated in Parkinson's disease through genome-wide association studies. However, GAK's role in mammalian neurons remains unclear, and insight may come from identification of further substrates. Employing a chemical genetics method, we show here that the sodium potassium pump (Na+/K+-ATPase) α-subunit Atp1a3 is a GAK target and that GAK regulates Na+/K+-ATPase trafficking to the plasma membrane. Whole-cell patch clamp recordings from CA1 pyramidal neurons in GAK conditional knockout mice show a larger change in resting membrane potential when exposed to the Na+/K+-ATPase blocker ouabain, indicating compromised Na+/K+-ATPase function in GAK knockouts. Our results suggest a modulatory role for GAK via phosphoregulation of substrates such as Atp1a3 during cargo trafficking.

Detection of parathyroid hormone-like hormone in cancer cell cultures by gold nanoparticle-based lateral flow immunoassays.

Parathyroid hormone-like hormone (PTHLH) exerts relevant roles in progression and dissemination of several tumors. However, factors influencing its production and secretion have not been fully characterized. The main limitation is the lack of specific, sensitive and widely available techniques to detect and quantify PTHLH. We have developed a lateral flow immunoassay using gold nanoparticles label for the fast and easy detection of PTHLH in lysates and culture media of three human cell lines (HaCaT, LA-N-1, SK-N-AS). Levels in culture media and lysates ranged from 11 to 20 ng/mL and 0.66 to 0.87 µg/mL respectively. Results for HaCaT are in agreement to the previously reported, whereas LA-N-1 and SK-N-AS have been evaluated for the first time. The system also exhibits good performance in human serum samples. This methodology represents a helpful tool for future in vitro and in vivo studies of mechanisms involved in PTHLH production as well as for diagnostics. From the Clinical Editor: Parathyroid Hormone-like Hormone (PTHLH) is known to be secreted by some tumors. However, the detection of this peptide remains difficult. The authors here described their technique of using gold nanoparticles as label for the detection of PTHLH by Lateral-flow immunoassays (LFIAs). The positive results may also point a way to using the same technique for the rapid determination of other relevant cancer proteins.

Nanochannel array device operating through Prussian blue nanoparticles for sensitive label-free immunodetection of a cancer biomarker.

A novel nanochannel array (NC) device that operates through Prussian blue nanoparticles (PBNPs) as redox indicator for sensitive label free immunodetection of a cancer biomarker is presented. Stable and narrow-sized (around 4 nm) PBNPs, protected by polyvinylpyrrolidone, exhibited a well-defined and reproducible redox behavior and were successfully applied for the voltammetric evaluation of the nanochannels (20 nm pore sized) blockage due to the immunocomplex formation. The bigger size of the PBNPs compared with ionic indicators such as the [Fe(CN)6](4-/3-) system leads to an increase in the steric effects hindering their diffusion toward the signaling electrode which in turn is transduced to an improvement of the detection limit from 200 µg mL(-1) to 34 pg human IgG mL(-1). This novel and effective PBNPs-NC technology for the detection of small proteins captured inside the nanochannels is successfully applied for the quantification of a cancer biomarker (parathyroid hormone-related protein, PTHrP) in a real clinical scenario such as cell culture medium. The achieved label-free detection of PTHrP at levels of 50 ng mL(-1) is with great interest to study relevant functions that this protein exerts in normal tissues and cancer.

Pituitary adenylate cyclase-activating polypeptide (PACAP) inhibits the slow afterhyperpolarizing current sIAHP in CA1 pyramidal neurons by activating multiple signaling pathways.

The slow afterhyperpolarizing current (sIAHP ) is a calcium-dependent potassium current that underlies the late phase of spike frequency adaptation in hippocampal and neocortical neurons. sIAHP is a well-known target of modulation by several neurotransmitters acting via the cyclic AMP (cAMP) and protein kinase A (PKA)-dependent pathway. The neuropeptide pituitary adenylate cyclase activating peptide (PACAP) and its receptors are present in the hippocampal formation. In this study we have investigated the effect of PACAP on the sIAHP and the signal transduction pathway used to modulate intrinsic excitability of hippocampal pyramidal neurons. We show that PACAP inhibits the sIAHP , resulting in a decrease of spike frequency adaptation, in rat CA1 pyramidal cells. The suppression of sIAHP by PACAP is mediated by PAC1 and VPAC1 receptors. Inhibition of PKA reduced the effect of PACAP on sIAHP, suggesting that PACAP exerts part of its inhibitory effect on sIAHP by increasing cAMP and activating PKA. The suppression of sIAHP by PACAP was also strongly hindered by the inhibition of p38 MAP kinase (p38 MAPK). Concomitant inhibition of PKA and p38 MAPK indicates that these two kinases act in a sequential manner in the same pathway leading to the suppression of sIAHP. Conversely, protein kinase C is not part of the signal transduction pathway used by PACAP to inhibit sIAHP in CA1 neurons. Our results show that PACAP enhances the excitability of CA1 pyramidal neurons by inhibiting the sIAHP through the activation of multiple signaling pathways, most prominently cAMP/PKA and p38 MAPK. Our findings disclose a novel modulatory action of p38 MAPK on intrinsic excitability and the sIAHP, underscoring the role of this current as a neuromodulatory hub regulated by multiple protein kinases in cortical neurons.

Casein modified gold nanoparticles for future theranostic applications.

The synthesis and characterization of gold nanoparticles (AuNPs 20 nm sized) modified with k-casein derived peptides in order to monitor the peptide effect as bacterial adhesion inhibitor, thanks to the carrier/concentrator effect of the AuNPs is here presented. Some aspects related to the stability of AuNP/peptide conjugates for a potential application in the design of an electrochemical biosensor for pathogen bacteria detection are also discussed. This peptide based nanoparticle assay takes advantage of the dual character of the AuNPs: as carrier of the biorecognition molecule and also as electrocatalytic label, allowing the evaluation of the pathogen bacteria-peptide interaction in a simple and rapid way through the chronoamperometric monitoring of the hydrogen evolution reaction on screen-printed carbon electrodes. The developed proof of concept theranostic system may open the way to therapeutic and biosensing applications with interest for various fields.

Small-conductance Ca2+-activated K+ channels modulate action potential-induced Ca2+ transients in hippocampal neurons.

In hippocampal pyramidal neurons, voltage-gated Ca(2+) channels open in response to action potentials. This results in elevations in the intracellular concentration of Ca(2+) that are maximal in the proximal apical dendrites and decrease rapidly with distance from the soma. The control of these action potential-evoked Ca(2+) elevations is critical for the regulation of hippocampal neuronal activity. As part of Ca(2+) signaling microdomains, small-conductance Ca(2+)-activated K(+) (SK) channels have been shown to modulate the amplitude and duration of intracellular Ca(2+) signals by feedback regulation of synaptically activated Ca(2+) sources in small distal dendrites and dendritic spines, thus affecting synaptic plasticity in the hippocampus. In this study, we investigated the effect of the activation of SK channels on Ca(2+) transients specifically induced by action potentials in the proximal processes of hippocampal pyramidal neurons. Our results, obtained by using selective SK channel blockers and enhancers, show that SK channels act in a feedback loop, in which their activation by Ca(2+) entering mainly through L-type voltage-gated Ca(2+) channels leads to a reduction in the subsequent dendritic influx of Ca(2+). This underscores a new role of SK channels in the proximal apical dendrite of hippocampal pyramidal neurons.

Evaluación electrofisiológica de la memoria semántica en la enfermedad de Alzheimer / Electrophysiological assessment of semantic memory in Alzheimer's disease

El presente estudio utilizó la técnica de los Potenciales Relacionados a Eventos (PREs) para analizar la actividad eléctrica cerebral de un grupo de diez pacientes con enfermedad de Alzheimer (EA) y un grupo de 10 ancianos asintomáticos ante una prueba de categorización semántica. La tarea se conformó de 118 parejas de dibujos de objetos y animales comunes. El 50 por ciento de las parejas pertenecían a un mismo grupo supraordinado y constituía la condición congruente (ej. guitarra-violín), el otro 50 por ciento pertenecía a diferentes grupos supraordinados y constituía la condición incongruente (ej. casco-violín). Al comparar los PREs de ambos grupos, se observó una reducción significativa del componente N400 en los pacientes con EA. Esta reducción se debió a una pronunciada positividad de la actividad eléctrica cerebral ante la condición incongruente. La disminución del N400 en los PREs de los pacientes con EA apoya la hipótesis sobre la existencia de dificultades para acceder y utilizar la información semántica de manera eficiente