Publications by authors named "Francesco Miceli"

49 Publications

Editorial: Kv7 Channels: Structure, Physiology, and Pharmacology.

Front Physiol 2021 16;12:679317. Epub 2021 Apr 16.

Department of Neuroscience, Reproductive Sciences and Dentistry, University of Naples Federico II, Naples, Italy.

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http://dx.doi.org/10.3389/fphys.2021.679317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8085343PMC
April 2021

Generation of an iPSC line (UNINAi001-A) from a girl with neonatal-onset epilepsy and non-syndromic intellectual disability carrying the homozygous KCNQ3 p.PHE534ILEfs*15 variant and of an iPSC line (UNINAi002-A) from a non-carrier, unaffected brother.

Stem Cell Res 2021 Mar 24;53:102311. Epub 2021 Mar 24.

Departments of Neuroscience and Molecular Medicine and Medical Biotechnology, University bf Naples "Federico II", Naples, Italy.

Heterozygous variants in the KCNQ3 gene cause epileptic and/or developmental disorders of varying severity. Here we describe the generation of induced pluripotent stem cells (iPSCs) from a 9-year-old girl with pharmacodependent neonatal-onset epilepsy and intellectual disability who carry a homozygous single-base duplication in exon 12 of KCNQ3 (NM_004519.3: KCNQ3 c.1599dup; KCNQ3 p.PHE534ILEfs*15), and from a non-carrier brother of the proband. For iPSC generation, non-integrating episomal plasmid vectors were used to transfect fibroblasts isolated from skin biopsies. The obtained iPSC lines had a normal karyotype, showed embryonic stem cell-like morphology, expressed pluripotency markers, and possessed trilineage differentiation potential.
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http://dx.doi.org/10.1016/j.scr.2021.102311DOI Listing
March 2021

Genotype-phenotype correlations in patients with de novo pathogenic variants.

Neurol Genet 2020 Dec 30;6(6):e528. Epub 2020 Nov 30.

Department of Neurosciences (F. Malerba, G.B., E.A., A. Riva, V.S., L.N., C. Minetti, F.Z., P.S.), Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health, Università degli Studi di Genova; Pediatric Neurology and Muscular Diseases Unit (F. Malerba, G.B., F. Marchese, E.A., A. Riva, M.S.V., V.S., C. Minetti, P.S.), IRCCS Istituto G. Gaslini; Center for Synaptic Neuroscience and Technology (NSYN@UniGe) (G.A., L.M., F.B.), Istituto Italiano di Tecnologia; Department of Experimental Medicine (G.A.), Università degli Studi di Genova; Laboratory of Human Genetics (E.G.); Unit of Medical Genetics (F. Madia, F.Z.), IRCCS Istituto G. Gaslini, Genova, Italy; Child Neurology and Neurorehabilitation Unit (M.A.), Department of Pediatrics, Central Hospital of Bolzano, Bolzano; Child Neurology and Psychiatry Unit (L.G., P.A., P.M.), ASST Spedali Civili, Brescia; Neurology Unit (M. Trivisano, N.S.), Department of Neuroscience, Bambino Gesù Children's Hospital, IRCCS, Roma; Child Neurology Unit (A. Russo, G.G.), IRCCS, Institute of Neurological Sciences of Bologna; Child Neuropsychiatry Unit (F.R.), U.O.N.P.I.A. ASST-Rhodense, Rho, Milano; Neurology Unit and Laboratories (T.P.), A. Meyer Children's Hospital, Firenze; Child Neurology and Psychiatric Unit (C. Marini), Pediatric Hospital G. Salesi, United Hospital of Ancona; Child Neuropsychiatry Unit (M.M.M., L.N.), IRCCS Istituto G. Gaslini, Genova; Department of Pediatric Neuroscience (E.F.), Fondazione IRCCS Istituto Neurologico Carlo Besta; Unit of Genetics of Neurodegenerative and Metabolic Diseases (B. Castellotti), Fondazione IRCCS Istituto Neurologico Carlo Besta, Milano; Department of Child Neuropsychiatry (G.C.), Epilepsy Center, C. Poma Hospital, Mantova; Fondazione Poliambulanza Brescia (G.C.); Epilepsy Center (A.C.), Department of Neuroscience, Reproductive and Odontostomatological Sciences, Università degli Studi di Napoli Federico II, Napoli; Department of Pediatrics (A.V.), University of Perugia; Section of Pharmacology (F. Miceli, M. Taglialatela), Department of Neuroscience, Reproductive and Odontostomatological Sciences, Università degli Studi di Napoli Federico II, Napoli; IRCCS Ospedale Policlinico San Martino (L.M., F.B.), Genova, Italy; Division of Pediatric Neurology (M.R.C.), Saint-Luc University Hospital, and Institute of Experimental and Clinical Research (IREC), Université Catholique de Louvain, Brussels, Belgium; Department of Epilepsy Genetics and Personalized Treatment (K.M.J., R.S.M.), The Danish Epilepsy Center Filadelfia, Dianalund, Denmark; Institute for Regional Health Services (K.M.J., R.S.M.), University of Southern Denmark, Odense, Denmark; Department of Neurology (B. Ceulemans, S.W.), University Hospital Antwerp; Applied & Translational Neurogenomics Group (S.W.), VIB-Center for Molecular Neurology; Laboratory of Neurogenetics (S.W.), Institute Born-Bunge, University of Antwerp, Belgium; and Department of Life and Environmental Sciences (L.M.), Polytechnic University of Marche, Ancona, Italy.

Objective: Early identification of de novo variants in patients with epilepsy raises prognostic issues toward optimal management. We analyzed the clinical and genetic information from a cohort of patients with de novo pathogenic variants to dissect genotype-phenotype correlations.

Methods: Patients with de novo pathogenic variants were identified from Italy, Denmark, and Belgium. Atomic resolution Kv7.2 structures were also generated using homology modeling to map the variants.

Results: We included 34 patients with a mean age of 4.7 years. Median seizure onset was 2 days, mainly with focal seizures with autonomic signs. Twenty-two patients (65%) were seizure free at the mean age of 1.2 years. More than half of the patients (17/32) displayed severe/profound intellectual disability; however, 4 (13%) of them had a normal cognitive outcome.A total of 28 de novo pathogenic variants were identified, most missense (25/28), and clustered in conserved regions of the protein; 6 variants recurred, and 7 were novel. We did not identify a relationship between variant position and seizure offset or cognitive outcome in patients harboring missense variants. Besides, recurrent variants were associated with overlapping epilepsy features but also variable evolution regarding the intellectual outcome.

Conclusions: We highlight the complexity of variant interpretation to assess the impact of a class of de novo mutations. Genetic modifiers could be implicated, but the study paradigms to successfully address the impact of each single mutation need to be developed.
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http://dx.doi.org/10.1212/NXG.0000000000000528DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7803337PMC
December 2020

Benign familial infantile epilepsy associated with KCNQ3 mutation: a rare occurrence or an underestimated event?

Epileptic Disord 2020 Dec;22(6):807-810

Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK.

Benign familial infantile epilepsy (BFIE) is the most genetically heterogeneous phenotype among early-onset familial infantile epilepsies. It has an autosomal dominant inheritance pattern with incomplete penetrance. Although PRRT2 is the most mutated gene detected in families with BFIE, other mutations in KCNQ2, SCN2A, and GABRA6 genes have also been described. To date, KCNQ3 mutations have been detected in only four patients with BFIE. Here, we describe the clinical pattern and course of an additional individual with BFIE associated with a novel missense heterozygous KCNQ3 c.1850G>C variant inherited by his unaffected father. The incidence of KCNQ3 mutations among BFIE patients is reported to be low in the literature, however, whether this is underestimated is unclear as not all current epilepsy gene panels include KCNQ3.
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http://dx.doi.org/10.1684/epd.2020.1221DOI Listing
December 2020

The Role of Kv7.2 in Neurodevelopment: Insights and Gaps in Our Understanding.

Front Physiol 2020 28;11:570588. Epub 2020 Oct 28.

Applied and Translational Neurogenomics Group, VIB Center for Molecular Neurology, Vlaams Instituut voor Biotechnologie, Antwerp, Belgium.

Kv7.2 subunits encoded by the gene constitute a critical molecular component of the M-current, a subthreshold voltage-gated potassium current controlling neuronal excitability by dampening repetitive action potential firing. Pathogenic loss-of-function variants in have been linked to epilepsy since 1998, and there is ample functional evidence showing that dysfunction of the channel indeed results in neuronal hyperexcitability. The recent description of individuals with severe developmental delay with or without seizures due to pathogenic variants in (-encephalopathy) reveals that Kv7.2 channels also have an important role in neurodevelopment. Kv7.2 channels are expressed already very early in the developing brain when key developmental processes such as proliferation, differentiation, and synaptogenesis play a crucial role in brain morphogenesis and maturation. In this review, we will discuss the available evidence for a role of Kv7.2 channels in these neurodevelopmental processes, focusing in particular on insights derived from -related human phenotypes, from the spatio-temporal expression of Kv7.2 and other Kv7 family member, and from cellular and rodent models, highlighting critical gaps and research strategies to be implemented in the future. Lastly, we propose a model which divides the M-current activity in three different developmental stages, correlating with the cell characteristics during these particular periods in neuronal development, and how this can be linked with -related disorders. Understanding these mechanisms can create opportunities for new targeted therapies for -encephalopathy.
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http://dx.doi.org/10.3389/fphys.2020.570588DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7657400PMC
October 2020

A Novel Kv7.3 Variant in the Voltage-Sensing S Segment in a Family With Benign Neonatal Epilepsy: Functional Characterization and Rescue by β-Hydroxybutyrate.

Front Physiol 2020 4;11:1040. Epub 2020 Sep 4.

Institute for Genomic Medicine, Columbia University Irving Medical Center, New York, NY, United States.

Pathogenic variants in and , paralogous genes encoding Kv7.2 and Kv7.3 voltage-gated K channel subunits, are responsible for early-onset developmental/epileptic disorders characterized by heterogeneous clinical phenotypes ranging from benign familial neonatal epilepsy (BFNE) to early-onset developmental and epileptic encephalopathy (DEE). variants account for the majority of pedigrees with BFNE and variants are responsible for a much smaller subgroup, but the reasons for this imbalance remain unclear. Analysis of additional pedigrees is needed to further clarify the nature of this genetic heterogeneity and to improve prediction of pathogenicity for novel variants. We identified a BFNE family with two siblings and a parent affected. Exome sequencing on samples from both parents and siblings revealed a novel variant (c.719T>G; p.M240R), segregating in the three affected individuals. The M240 residue is conserved among human Kv7.2-5 and lies between the two arginines (R5 and R6) closest to the intracellular side of the voltage-sensing S transmembrane segment. Whole cell patch-clamp recordings in Chinese hamster ovary (CHO) cells revealed that homomeric Kv7.3 M240R channels were not functional, whereas heteromeric channels incorporating Kv7.3 M240R mutant subunits with Kv7.2 and Kv7.3 displayed a depolarizing shift of about 10 mV in activation gating. Molecular modeling results suggested that the M240R substitution preferentially stabilized the resting state and possibly destabilized the activated state of the Kv7.3 subunits, a result consistent with functional data. Exposure to β-hydroxybutyrate (BHB), a ketone body generated during the ketogenic diet (KD), reversed channel dysfunction induced by the M240R variant. In conclusion, we describe the first missense loss-of-function (LoF) pathogenic variant within the S segment of Kv7.3 identified in patients with BFNE. Studied under conditions mimicking heterozygosity, the M240R variant mainly affects the voltage sensitivity, in contrast to previously analyzed BFNE Kv7.3 variants that reduce current density. Our pharmacological results provide a rationale for the use of KD in patients carrying LoF variants in Kv7.2 or Kv7.3 subunits.
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http://dx.doi.org/10.3389/fphys.2020.01040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7498716PMC
September 2020

Cytokine storm in aged people with CoV-2: possible role of vitamins as therapy or preventive strategy.

Aging Clin Exp Res 2020 Oct 31;32(10):2115-2131. Epub 2020 Aug 31.

Department of Pharmacy and Biotechnology (FABIT), University of Bologna, Bologna, Italy.

Background: In December 2019, a novel human-infecting coronavirus, SARS-CoV-2, had emerged. The WHO has classified the epidemic as a "public health emergency of international concern". A dramatic situation has unfolded with thousands of deaths, occurring mainly in the aged and very ill people. Epidemiological studies suggest that immune system function is impaired in elderly individuals and these subjects often present a deficiency in fat-soluble and hydrosoluble vitamins.

Methods: We searched for reviews describing the characteristics of autoimmune diseases and the available therapeutic protocols for their treatment. We set them as a paradigm with the purpose to uncover common pathogenetic mechanisms between these pathological conditions and SARS-CoV-2 infection. Furthermore, we searched for studies describing the possible efficacy of vitamins A, D, E, and C in improving the immune system function.

Results: SARS-CoV-2 infection induces strong immune system dysfunction characterized by the development of an intense proinflammatory response in the host, and the development of a life-threatening condition defined as cytokine release syndrome (CRS). This leads to acute respiratory syndrome (ARDS), mainly in aged people. High mortality and lethality rates have been observed in elderly subjects with CoV-2-related infection.

Conclusions: Vitamins may shift the proinflammatory Th17-mediated immune response arising in autoimmune diseases towards a T-cell regulatory phenotype. This review discusses the possible activity of vitamins A, D, E, and C in restoring normal antiviral immune system function and the potential therapeutic role of these micronutrients as part of a therapeutic strategy against SARS-CoV-2 infection.
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http://dx.doi.org/10.1007/s40520-020-01669-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7456763PMC
October 2020

Epileptic channelopathies caused by neuronal Kv7 (KCNQ) channel dysfunction.

Pflugers Arch 2020 07 6;472(7):881-898. Epub 2020 Jun 6.

Section of Pharmacology, Department of Neuroscience, University of Naples, "Federico II", Via Pansini 5, 80131, Naples, Italy.

Seizures are the most common neurological manifestation in the newborn period, with an estimated incidence of 1.8-3.5 per 1000 live births. Prolonged or intractable seizures have a detrimental effect on cognition and brain function in experimental animals and are associated with adverse long-term neurodevelopmental sequelae and an increased risk of post-neonatal epilepsy in humans. The developing brain is particularly susceptible to the potentially severe effects of epilepsy, and epilepsy, especially when refractory to medications, often results in a developmental and epileptic encephalopathy (DEE) with developmental arrest or regression. DEEs can be primarily attributed to genetic causes. Given the critical role of potassium (K) currents with distinct subcellular localization, biophysical properties, modulation, and pharmacological profile in regulating intrinsic electrical properties of neurons and their responsiveness to synaptic inputs, it is not too surprising that genetic research in the past two decades has identified several K channel genes as responsible for a large fraction of DEE. In the present article, we review the genetically determined epileptic channelopathies affecting three members of the Kv7 family, namely Kv7.2 (KCNQ2), Kv7.3 (KCNQ3), and Kv7.5 (KCNQ5); we review the phenotypic spectrum of Kv7-related epileptic channelopathies, the different genetic and pathogenetic mechanisms, and the emerging genotype-phenotype correlations which may prove crucial for prognostic predictions, disease management, parental counseling, and individually tailored therapeutic attempts.
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http://dx.doi.org/10.1007/s00424-020-02404-2DOI Listing
July 2020

Synthesis and Pharmacological Characterization of Conformationally Restricted Retigabine Analogues as Novel Neuronal Kv7 Channel Activators.

J Med Chem 2020 01 24;63(1):163-185. Epub 2019 Dec 24.

Department of Neuroscience, Reproductive Sciences and Dentistry , University Federico II of Naples , Via Pansini, 5 , 80131 Naples , Italy.

Kv7 K channels represent attractive pharmacological targets for the treatment of different neurological disorders, including epilepsy. In this paper, 42 conformationally restricted analogues of the prototypical Kv7 activator retigabine have been synthesized and tested by electrophysiological patch-clamp experiments as Kv7 agonists. When compared to retigabine (0.93 ± 0.43 μM), the ECs for Kv7.2 current enhancements by compound (0.08 ± 0.04 μM) were lower, whereas no change in potency was observed for (0.63 ± 0.07 μM). In addition, compared to retigabine, and showed also higher potency in activating heteromeric Kv7.2/Kv7.3 and homomeric Kv7.4 channels. Molecular modeling studies provided new insights into the chemical features required for optimal interaction at the binding site. Stability studies evidenced improved chemical stability of and in comparison with retigabine. Overall, the present results highlight that the 5-alkylamidoindole moiety provides a suitable pharmacophoric scaffold for the design of chemically stable, highly potent and selective Kv7 agonists.
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http://dx.doi.org/10.1021/acs.jmedchem.9b00796DOI Listing
January 2020

Insights into the pathogenesis of ATP1A1-related CMT disease using patient-specific iPSCs.

J Peripher Nerv Syst 2019 12 24;24(4):330-339. Epub 2019 Nov 24.

Department of Neuroscience, Reproductive Sciences and Odontostomatology, University of Naples "Federico II", Naples, Italy.

The development of patient-specific induced pluripotent stem cells (iPSCs) offered interesting insights in modeling the pathogenesis of Charcot-Marie-Tooth (CMT) disease and thus we decided to explore the phenotypes of iPSCs derived from a single CMT patient carrying a mutant ATP1A1 allele (p.Pro600Ala). iPSCs clones generated from CMT and control fibroblasts, were induced to differentiate into neural precursors and then into post-mitotic neurons. Control iPSCs differentiated into neuronal precursors and then into post-mitotic neurons within 6-8 days. On the contrary, the differentiation of CMT iPSCs was clearly defective. Electrophysiological properties confirmed that post-mitotic neurons were less mature compared to the normal counterpart. The impairment of in vitro differentiation of CMT iPSCs only concerned with the neuronal pathway, because they were able to differentiate into mesendodermal cells and other ectodermal derivatives. ATP1A1 was undetectable in the few neuronal cells derived from CMT iPSCs. ATP1A1 gene mutation (p.Pro600Ala), responsible for a form of axonal CMT disease, is associated in vitro with a dramatic alteration of the differentiation of patient-derived iPSCs into post-mitotic neurons. Thus, the defect in neuronal cell development might lead in vivo to a decreased number of mature neurons in ATP1A1-CMT disease.
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http://dx.doi.org/10.1111/jns.12357DOI Listing
December 2019

Activation of Kv7 Potassium Channels Inhibits Intracellular Ca Increases Triggered By TRPV1-Mediated Pain-Inducing Stimuli in F11 Immortalized Sensory Neurons.

Int J Mol Sci 2019 Sep 4;20(18). Epub 2019 Sep 4.

Division of Pharmacology, Department of Neuroscience, University of Naples "Federico II", 80131 Naples, Italy.

Kv7.2-Kv7.5 channels mediate the M-current (I), a K-selective current regulating neuronal excitability and representing an attractive target for pharmacological therapy against hyperexcitability diseases such as pain. Kv7 channels interact functionally with transient receptor potential vanilloid 1 (TRPV1) channels activated by endogenous and/or exogenous pain-inducing substances, such as bradykinin (BK) or capsaicin (CAP), respectively; however, whether Kv7 channels of specific molecular composition provide a dominant contribution in BK- or CAP-evoked responses is yet unknown. To this aim, Kv7 transcripts expression and function were assessed in F11 immortalized sensorial neurons, a cellular model widely used to assess nociceptive molecular mechanisms. In these cells, the effects of the pan-Kv7 activator retigabine were investigated, as well as the effects of ICA-27243 and (S)-1, two Kv7 activators acting preferentially on Kv7.2/Kv7.3 and Kv7.4/Kv7.5 channels, respectively, on BK- and CAP-induced changes in intracellular Ca concentrations ([Ca]). The results obtained revealed the expression of transcripts of all genes, leading to an I-like current. Moreover, all tested Kv7 openers inhibited BK- and CAP-induced responses by a similar extent (~60%); at least for BK-induced Ca responses, the potency of retigabine (IC~1 µM) was higher than that of ICA-27243 (IC~5 µM) and (S)-1 (IC~7 µM). Altogether, these results suggest that I activation effectively counteracts the cellular processes triggered by TRPV1-mediated pain-inducing stimuli, and highlight a possible critical contribution of Kv7.4 subunits.
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http://dx.doi.org/10.3390/ijms20184322DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6769798PMC
September 2019

A novel homozygous KCNQ3 loss-of-function variant causes non-syndromic intellectual disability and neonatal-onset pharmacodependent epilepsy.

Epilepsia Open 2019 Sep 11;4(3):464-475. Epub 2019 Aug 11.

Division of Pharmacology, Department of Neuroscience University of Naples "Federico II" Naples Italy.

Objective: Heterozygous variants in or, more rarely, genes are responsible for early-onset developmental/epileptic disorders characterized by heterogeneous clinical presentation and course, genetic transmission, and prognosis. While familial forms mostly include benign epilepsies with seizures starting in the neonatal or early-infantile period, de novo variants in or have been described in sporadic cases of early-onset encephalopathy (EOEE) with pharmacoresistant seizures, various age-related pathological EEG patterns, and moderate/severe developmental impairment. All pathogenic variants in or occur in heterozygosity. The aim of this work was to report the clinical, molecular, and functional properties of a new variant found in homozygous configuration in a 9-year-old girl with pharmacodependent neonatal-onset epilepsy and non-syndromic intellectual disability.

Methods: Exome sequencing was used for genetic investigation. KCNQ3 transcript and subunit expression in fibroblasts was analyzed with quantitative real-time PCR and Western blotting or immunofluorescence, respectively. Whole-cell patch-clamp electrophysiology was used for functional characterization of mutant subunits.

Results: A novel single-base duplication in exon 12 of (NM_004519.3:c.1599dup) was found in homozygous configuration in the proband born to consanguineous healthy parents; this frameshift variant introduced a premature termination codon (PTC), thus deleting a large part of the C-terminal region. Mutant KCNQ3 transcript and protein abundance was markedly reduced in primary fibroblasts from the proband, consistent with nonsense-mediated mRNA decay. The variant fully abolished the ability of KCNQ3 subunits to assemble into functional homomeric or heteromeric channels with KCNQ2 subunits.

Significance: The present results indicate that a homozygous loss-of-function variant is responsible for a severe phenotype characterized by neonatal-onset pharmacodependent seizures, with developmental delay and intellectual disability. They also reveal difference in genetic and pathogenetic mechanisms between - and -related epilepsies, a crucial observation for patients affected with EOEE and/or developmental disabilities.
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http://dx.doi.org/10.1002/epi4.12353DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6698674PMC
September 2019

Epileptic Encephalopathy In A Patient With A Novel Variant In The Kv7.2 S2 Transmembrane Segment: Clinical, Genetic, and Functional Features.

Int J Mol Sci 2019 Jul 10;20(14). Epub 2019 Jul 10.

Department of Neuroscience, University of Naples "Federico II", 80131 Naples, Italy.

Kv7.2 subunits encoded by the gene provide a major contribution to the M-current (I), a voltage-gated K current crucially involved in the regulation of neuronal excitability. Heterozygous missense variants in Kv7.2 are responsible for epileptic diseases characterized by highly heterogeneous genetic transmission and clinical severity, ranging from autosomal-dominant Benign Familial Neonatal Seizures (BFNS) to sporadic cases of severe epileptic and developmental encephalopathy (DEE). Here, we describe a patient with neonatal onset DEE, carrying a previously undescribed heterozygous c.418G > C, p.Glu140Gln (E140Q) variant. Patch-clamp recordings in CHO cells expressing the E140Q mutation reveal dramatic loss of function (LoF) effects. Multistate structural modelling suggested that the E140Q substitution impeded an intrasubunit electrostatic interaction occurring between the E140 side chain in S and the arginine at position 210 in S (R210); this interaction is critically involved in stabilizing the activated configuration of the voltage-sensing domain (VSD) of Kv7.2. Functional results from coupled charge reversal or disulfide trapping experiments supported such a hypothesis. Finally, retigabine restored mutation-induced functional changes, reinforcing the rationale for the clinical use of Kv7 activators as personalized therapy for DEE-affected patients carrying Kv7.2 LoF mutations.
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http://dx.doi.org/10.3390/ijms20143382DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6678645PMC
July 2019

Autism and developmental disability caused by KCNQ3 gain-of-function variants.

Ann Neurol 2019 08 26;86(2):181-192. Epub 2019 Jun 26.

Division of Neurology, Department of Pediatrics, Children's Hospital of Pittsburgh and University of Pittsburgh School of Medicine, Pittsburgh, PA.

Objective: Recent reports have described single individuals with neurodevelopmental disability (NDD) harboring heterozygous KCNQ3 de novo variants (DNVs). We sought to assess whether pathogenic variants in KCNQ3 cause NDD and to elucidate the associated phenotype and molecular mechanisms.

Methods: Patients with NDD and KCNQ3 DNVs were identified through an international collaboration. Phenotypes were characterized by clinical assessment, review of charts, electroencephalographic (EEG) recordings, and parental interview. Functional consequences of variants were analyzed in vitro by patch-clamp recording.

Results: Eleven patients were assessed. They had recurrent heterozygous DNVs in KCNQ3 affecting residues R230 (R230C, R230H, R230S) and R227 (R227Q). All patients exhibited global developmental delay within the first 2 years of life. Most (8/11, 73%) were nonverbal or had a few words only. All patients had autistic features, and autism spectrum disorder (ASD) was diagnosed in 5 of 11 (45%). EEGs performed before 10 years of age revealed frequent sleep-activated multifocal epileptiform discharges in 8 of 11 (73%). For 6 of 9 (67%) recorded between 1.5 and 6 years of age, spikes became near-continuous during sleep. Interestingly, most patients (9/11, 82%) did not have seizures, and no patient had seizures in the neonatal period. Voltage-clamp recordings of the mutant KCNQ3 channels revealed gain-of-function (GoF) effects.

Interpretation: Specific GoF variants in KCNQ3 cause NDD, ASD, and abundant sleep-activated spikes. This new phenotype contrasts both with self-limited neonatal epilepsy due to KCNQ3 partial loss of function, and with the neonatal or infantile onset epileptic encephalopathies due to KCNQ2 GoF. ANN NEUROL 2019;86:181-192.
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http://dx.doi.org/10.1002/ana.25522DOI Listing
August 2019

Early Treatment with Quinidine in 2 Patients with Epilepsy of Infancy with Migrating Focal Seizures (EIMFS) Due to Gain-of-Function KCNT1 Mutations: Functional Studies, Clinical Responses, and Critical Issues for Personalized Therapy.

Neurotherapeutics 2018 10;15(4):1112-1126

Department of Medicine and Health Science, University of Molise, 86100, Campobasso, Italy.

Epilepsy of infancy with migrating focal seizures (EIMFS) is a rare early-onset developmental epileptic encephalopathy resistant to anti-epileptic drugs. The most common cause for EIMFS is a gain-of-function mutation in the KCNT1 potassium channel gene, and treatment with the KCNT1 blocker quinidine has been suggested as a rational approach for seizure control in EIMFS patients. However, variable results on the clinical efficacy of quinidine have been reported. In the present study, we provide a detailed description of the clinical, genetic, in vitro, and in vivo electrophysiological profile and pharmacological responses to quinidine of 2 EIMFS unrelated patients with a heterozygous de novo KCNT1 mutation: c.2849G>A (p.R950Q) in patient 1 and c.2677G>A (p.E893K) in patient 2. When expressed heterologously in CHO cells, KCNT1 channels carrying each variant showed gain-of-function effects, and were more effectively blocked by quinidine when compared to wild-type KCNT1 channels. On the basis of these in vitro results, add-on quinidine treatment was started at 3 and 16 months of age in patients 1 and 2, respectively. The results obtained reveal that quinidine significantly reduced seizure burden (by about 90%) and improved quality of life in both patients, but failed to normalize developmental milestones, which persisted as severely delayed. Based on the present experience, early quinidine intervention associated with heart monitoring and control of blood levels is among the critical factors for therapy effectiveness in EIMFS patients with KCNT1 gain-of-function mutations. Multicenter studies are needed to establish a consensus protocol for patient recruitment, quinidine treatment modalities, and outcome evaluation, to optimize clinical efficacy and reduce risks as well as variability associated to quinidine use in such severe developmental encephalopathy.
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http://dx.doi.org/10.1007/s13311-018-0657-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6277296PMC
October 2018

Kv7.3 Compound Heterozygous Variants in Early Onset Encephalopathy Reveal Additive Contribution of C-Terminal Residues to PIP-Dependent K Channel Gating.

Mol Neurobiol 2018 Aug 30;55(8):7009-7024. Epub 2018 Jan 30.

Department of Medicine and Health Science, University of Molise, Campobasso, Italy.

Over one hundred mutations in the Kv7.2 (KCNQ2) gene encoding for phosphatidylinositol 4,5-bisphosphate (PIP)-sensitive voltage-gated K channel subunits have been identified in early-onset epilepsies with wide phenotypic variability. By contrast, only few mutations in the closely related Kv7.3 (KCNQ3) gene have been reported, mostly associated with typical benign familial neonatal seizures (BFNS). We herein describe a patient affected by early onset epileptic encephalopathy (EOEE) carrying two Kv7.3 missense mutations (p.Val359Leu/V359L and p.Asp542Asn/D542N) in compound heterozygosis, each inherited from an asymptomatic parent. Patch-clamp recordings from transiently transfected CHO cells showed that, when incorporated in physiologically relevant Kv7.2 + Kv7.3 heteromeric channels, expression of Kv7.3 V359L or Kv7.3 D542N subunits failed to affect current density, whereas a significant decrease was instead observed when these mutant subunits were both simultaneously present. Modeling and functional experiments revealed that each variant decreased PIP-dependent current regulation, with additive effects when the two were co-expressed. Moreover, expression of Kv7.2 subunits carrying the D535N variant previously described in three sporadic EOEE cases prompted functional changes more dramatic when compared to those of the corresponding D542N variant in Kv7.3, but similar to those observed when both Kv7.3 V359L and Kv7.3 D542N subunits were expressed together. Finally, the Kv7 activator retigabine restored channel dysfunction induced by each Kv7.2 or Kv7.3 variant(s). These results provide a plausible molecular explanation for the apparent recessive inheritance of the phenotype in the family investigated, and a rational basis for personalized therapy with Kv7 channel activators in EOEE patients carrying loss-of-function mutations in Kv7.2 or Kv7.3.
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http://dx.doi.org/10.1007/s12035-018-0883-5DOI Listing
August 2018

Pharmacological Targeting of Neuronal Kv7.2/3 Channels: A Focus on Chemotypes and Receptor Sites.

Curr Med Chem 2018 ;25(23):2637-2660

Department of Neuroscience, University of Naples "Federico II", 80131 Naples, Italy.

Background: The Kv7 (KCNQ) subfamily of voltage-gated potassium channels consists of 5 members (Kv7.1-5) each showing characteristic tissue distribution and physiological roles. Given their functional heterogeneity, Kv7 channels represent important pharmacological targets for the development of new drugs for neuronal, cardiovascular and metabolic diseases.

Objective: In the present manuscript, we focus on describing the pharmacological relevance and potential therapeutic applications of drugs acting on neuronally-expressed Kv7.2/3 channels, placing particular emphasis on the different chemotypes, and highlighting their pharmacodynamic and, whenever possible, pharmacokinetic peculiarities.

Methods: The present work is based on an in-depth search of the currently available scientific literature, and on our own experience and knowledge in the field of neuronal Kv7 channel pharmacology. Space limitations impeded to describe the full pharmacological potential of Kv7 channels; thus, we have chosen to focus on neuronal channels composed of Kv7.2 and Kv7.3 subunits, and to mainly concentrate on their involvement in epilepsy.

Results: An astonishing heterogeneity in the molecular scaffolds exploitable to develop Kv7.2/3 modulators is evident, with important structural/functional peculiarities of distinct compound classes.

Conclusion: In the present work we have attempted to show the current status and growing potential of the Kv7 pharmacology field. We anticipate a bright future for the field, and express our hopes that the efforts herein reviewed will result in an improved treatment of hyperexcitability (or any other) diseases.
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http://dx.doi.org/10.2174/0929867324666171012122852DOI Listing
July 2018

Early-onset epileptic encephalopathy caused by a reduced sensitivity of Kv7.2 potassium channels to phosphatidylinositol 4,5-bisphosphate.

Sci Rep 2016 12 1;6:38167. Epub 2016 Dec 1.

Department of Medicine and Health Sciences, University of Molise, Campobasso, Italy.

Kv7.2 and Kv7.3 subunits underlie the M-current, a neuronal K current characterized by an absolute functional requirement for phosphatidylinositol 4,5-bisphosphate (PIP). Kv7.2 gene mutations cause early-onset neonatal seizures with heterogeneous clinical outcomes, ranging from self-limiting benign familial neonatal seizures to severe early-onset epileptic encephalopathy (Kv7.2-EE). In this study, the biochemical and functional consequences prompted by a recurrent variant (R325G) found independently in four individuals with severe forms of neonatal-onset EE have been investigated. Upon heterologous expression, homomeric Kv7.2 R325G channels were non-functional, despite biotin-capture in Western blots revealed normal plasma membrane subunit expression. Mutant subunits exerted dominant-negative effects when incorporated into heteromeric channels with Kv7.2 and/or Kv7.3 subunits. Increasing cellular PIP levels by co-expression of type 1γ PI(4)P5-kinase (PIP5K) partially recovered homomeric Kv7.2 R325G channel function. Currents carried by heteromeric channels incorporating Kv7.2 R325G subunits were more readily inhibited than wild-type channels upon activation of a voltage-sensitive phosphatase (VSP), and recovered more slowly upon VSP switch-off. These results reveal for the first time that a mutation-induced decrease in current sensitivity to PIP is the primary molecular defect responsible for Kv7.2-EE in individuals carrying the R325G variant, further expanding the range of pathogenetic mechanisms exploitable for personalized treatment of Kv7.2-related epilepsies.
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http://dx.doi.org/10.1038/srep38167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5131271PMC
December 2016

Infantile spasms and encephalopathy without preceding neonatal seizures caused by KCNQ2 R198Q, a gain-of-function variant.

Epilepsia 2017 01 9;58(1):e10-e15. Epub 2016 Nov 9.

Department of Neuroscience, University of Naples "Federico II", Naples, Italy.

Variants in KCNQ2 encoding for K 7.2 neuronal K channel subunits lead to a spectrum of neonatal-onset epilepsies, ranging from self-limiting forms to severe epileptic encephalopathy. Most KCNQ2 pathogenic variants cause loss-of-function, whereas few increase channel activity (gain-of-function). We herein provide evidence for a new phenotypic and functional profile in KCNQ2-related epilepsy: infantile spasms without prior neonatal seizures associated with a gain-of-function gene variant. With use of an international registry, we identified four unrelated patients with the same de novo heterozygous KCNQ2 c.593G>A, p.Arg198Gln (R198Q) variant. All were born at term and discharged home without seizures or concern of encephalopathy, but developed infantile spasms with hypsarrhythmia (or modified hypsarrhythmia) between the ages of 4 and 6 months. At last follow-up (ages 3-11 years), all patients were seizure-free and had severe developmental delay. In vitro experiments showed that Kv7.2 R198Q subunits shifted current activation gating to hyperpolarized potentials, indicative of gain-of-function; in neurons, K 7.2 and K 7.2 R198Q subunits similarly populated the axon initial segment, suggesting that gating changes rather than altered subcellular distribution contribute to disease molecular pathogenesis. We conclude that KCNQ2 R198Q is a model for a new subclass of KCNQ2 variants causing infantile spasms and encephalopathy, without preceding neonatal seizures. A PowerPoint slide summarizing this article is available for download in the Supporting Information section here.
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http://dx.doi.org/10.1111/epi.13601DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5219941PMC
January 2017

Personalized Stem Cell Therapy to Correct Corneal Defects Due to a Unique Homozygous-Heterozygous Mosaicism of Ectrodactyly-Ectodermal Dysplasia-Clefting Syndrome.

Stem Cells Transl Med 2016 Aug 5;5(8):1098-105. Epub 2016 May 5.

Fondazione Banca degli Occhi del Veneto, Venice, Italy Department of Molecular Medicine, University of Padua, Padua, Italy

Unlabelled: : Ectrodactyly-ectodermal dysplasia-clefting (EEC) syndrome is a rare autosomal dominant disease caused by mutations in the p63 gene. To date, approximately 40 different p63 mutations have been identified, all heterozygous. No definitive treatments are available to counteract and resolve the progressive corneal degeneration due to a premature aging of limbal epithelial stem cells. Here, we describe a unique case of a young female patient, aged 18 years, with EEC and corneal dysfunction, who was, surprisingly, homozygous for a novel and de novo R311K missense mutation in the p63 gene. A detailed analysis of the degree of somatic mosaicism in leukocytes from peripheral blood and oral mucosal epithelial stem cells (OMESCs) from biopsies of buccal mucosa showed that approximately 80% were homozygous mutant cells and 20% were heterozygous. Cytogenetic and molecular analyses excluded genomic alterations, thus suggesting a de novo mutation followed by an allelic gene conversion of the wild-type allele by de novo mutant allele as a possible mechanism to explain the homozygous condition. R311K-p63 OMESCs were expanded in vitro and heterozygous holoclones selected following clonal analysis. These R311K-p63 OMESCs were able to generate well-organized and stratified epithelia in vitro, resembling the features of healthy tissues. This study supports the rationale for the development of cultured autologous oral mucosal epithelial stem cell sheets obtained by selected heterozygous R311K-p63 stem cells, as an effective and personalized therapy for reconstructing the ocular surface of this unique case of EEC syndrome, thus bypassing gene therapy approaches.

Significance: This case demonstrates that in a somatic mosaicism context, a novel homozygous mutation in the p63 gene can arise as a consequence of an allelic gene conversion event, subsequent to a de novo mutation. The heterozygous mutant R311K-p63 stem cells can be isolated by means of clonal analysis and given their good regenerative capacity, they may be used to successfully correct the corneal defects present in this unique case of ectrodactyly-ectodermal dysplasia-clefting syndrome.
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http://dx.doi.org/10.5966/sctm.2015-0358DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4954457PMC
August 2016

Expression and function of Kv7.4 channels in rat cardiac mitochondria: possible targets for cardioprotection.

Cardiovasc Res 2016 May 29;110(1):40-50. Epub 2015 Dec 29.

Department of Neuroscience, Division of Pharmacology, University of Naples 'Federico II', Naples, Italy Department of Medicine and Health Science "Vincenzo Tiberio", University of Molise, Campobasso, Italy

Aims: Plasmalemmal Kv7.1 (KCNQ1) channels are critical players in cardiac excitability; however, little is known on the functional role of additional Kv7 family members (Kv7.2-5) in cardiac cells. In this work, the expression, function, cellular and subcellular localization, and potential cardioprotective role against anoxic-ischaemic cardiac injury of Kv7.4 channels have been investigated.

Methods And Results: Expression of Kv7.1 and Kv7.4 transcripts was found in rat heart tissue by quantitative polymerase chain reaction. Western blots detected Kv7.4 subunits in mitochondria from Kv7.4-transfected cells, H9c2 cardiomyoblasts, freshly isolated adult cardiomyocytes, and whole hearts. Immunofluorescence experiments revealed that Kv7.4 subunits co-localized with mitochondrial markers in cardiac cells, with ∼ 30-40% of cardiac mitochondria being labelled by Kv7.4 antibodies, a result also confirmed by immunogold electron microscopy experiments. In isolated cardiac (but not liver) mitochondria, retigabine (1-30 µM) and flupirtine (30 µM), two selective Kv7 activators, increased Tl(+) influx, depolarized the membrane potential, and inhibited calcium uptake; all these effects were antagonized by the Kv7 blocker XE991. In intact H9c2 cells, reducing Kv7.4 expression by RNA interference blunted retigabine-induced mitochondrial membrane depolarization; in these cells, retigabine decreased mitochondrial Ca(2+) levels and increased radical oxygen species production, both effects prevented by XE991. Finally, retigabine reduced cellular damage in H9c2 cells exposed to anoxia/re-oxygenation and largely prevented the functional and morphological changes triggered by global ischaemia/reperfusion (I/R) in Langendorff-perfused rat hearts.

Conclusion: Kv7.4 channels are present and functional in cardiac mitochondria; their activation exerts a significant cardioprotective role, making them potential therapeutic targets against I/R-induced cardiac injury.
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http://dx.doi.org/10.1093/cvr/cvv281DOI Listing
May 2016

Molecular pathophysiology and pharmacology of the voltage-sensing module of neuronal ion channels.

Front Cell Neurosci 2015 15;9:259. Epub 2015 Jul 15.

Department of Neuroscience, University of Naples Federico II Naples, Italy ; Department of Medicine and Health Sciences, University of Molise Campobasso, Italy.

Voltage-gated ion channels (VGICs) are membrane proteins that switch from a closed to open state in response to changes in membrane potential, thus enabling ion fluxes across the cell membranes. The mechanism that regulate the structural rearrangements occurring in VGICs in response to changes in membrane potential still remains one of the most challenging topic of modern biophysics. Na(+), Ca(2+) and K(+) voltage-gated channels are structurally formed by the assembly of four similar domains, each comprising six transmembrane segments. Each domain can be divided into two main regions: the Pore Module (PM) and the Voltage-Sensing Module (VSM). The PM (helices S5 and S6 and intervening linker) is responsible for gate opening and ion selectivity; by contrast, the VSM, comprising the first four transmembrane helices (S1-S4), undergoes the first conformational changes in response to membrane voltage variations. In particular, the S4 segment of each domain, which contains several positively charged residues interspersed with hydrophobic amino acids, is located within the membrane electric field and plays an essential role in voltage sensing. In neurons, specific gating properties of each channel subtype underlie a variety of biological events, ranging from the generation and propagation of electrical impulses, to the secretion of neurotransmitters and to the regulation of gene expression. Given the important functional role played by the VSM in neuronal VGICs, it is not surprising that various VSM mutations affecting the gating process of these channels are responsible for human diseases, and that compounds acting on the VSM have emerged as important investigational tools with great therapeutic potential. In the present review we will briefly describe the most recent discoveries concerning how the VSM exerts its function, how genetically inherited diseases caused by mutations occurring in the VSM affects gating in VGICs, and how several classes of drugs and toxins selectively target the VSM.
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http://dx.doi.org/10.3389/fncel.2015.00259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4502356PMC
August 2015

Early-onset epileptic encephalopathy caused by gain-of-function mutations in the voltage sensor of Kv7.2 and Kv7.3 potassium channel subunits.

J Neurosci 2015 Mar;35(9):3782-93

Department of Neuroscience, University of Naples Federico II, 80131 Naples, Italy, Department of Medicine and Health Science, University of Molise, 86100 Campobasso, Italy, and

Mutations in Kv7.2 (KCNQ2) and Kv7.3 (KCNQ3) genes, encoding for voltage-gated K(+) channel subunits underlying the neuronal M-current, have been associated with a wide spectrum of early-onset epileptic disorders ranging from benign familial neonatal seizures to severe epileptic encephalopathies. The aim of the present work has been to investigate the molecular mechanisms of channel dysfunction caused by voltage-sensing domain mutations in Kv7.2 (R144Q, R201C, and R201H) or Kv7.3 (R230C) recently found in patients with epileptic encephalopathies and/or intellectual disability. Electrophysiological studies in mammalian cells transfected with human Kv7.2 and/or Kv7.3 cDNAs revealed that each of these four mutations stabilized the activated state of the channel, thereby producing gain-of-function effects, which are opposite to the loss-of-function effects produced by previously found mutations. Multistate structural modeling revealed that the R201 residue in Kv7.2, corresponding to R230 in Kv7.3, stabilized the resting and nearby voltage-sensing domain states by forming an intricate network of electrostatic interactions with neighboring negatively charged residues, a result also confirmed by disulfide trapping experiments. Using a realistic model of a feedforward inhibitory microcircuit in the hippocampal CA1 region, an increased excitability of pyramidal neurons was found upon incorporation of the experimentally defined parameters for mutant M-current, suggesting that changes in network interactions rather than in intrinsic cell properties may be responsible for the neuronal hyperexcitability by these gain-of-function mutations. Together, the present results suggest that gain-of-function mutations in Kv7.2/3 currents may cause human epilepsy with a severe clinical course, thus revealing a previously unexplored level of complexity in disease pathogenetic mechanisms.
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http://dx.doi.org/10.1523/JNEUROSCI.4423-14.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6605567PMC
March 2015

A novel KCNQ3 mutation in familial epilepsy with focal seizures and intellectual disability.

Epilepsia 2015 Feb 19;56(2):e15-20. Epub 2014 Dec 19.

Unit of Pharmacology, Department of Neuroscience, Reproductive Science and Dentistry, University of Naples Federico II, Naples, Italy.

Mutations in the KCNQ2 gene encoding for voltage-gated potassium channel subunits have been found in patients affected with early onset epilepsies with wide phenotypic heterogeneity, ranging from benign familial neonatal seizures (BFNS) to epileptic encephalopathy with cognitive impairment, drug resistance, and characteristic electroencephalography (EEG) and neuroradiologic features. By contrast, only few KCNQ3 mutations have been rarely described, mostly in patients with typical BFNS. We report clinical, genetic, and functional data from a family in which early onset epilepsy and neurocognitive deficits segregated with a novel mutation in KCNQ3 (c.989G>T; p.R330L). Electrophysiological studies in mammalian cells revealed that incorporation of KCNQ3 R330L mutant subunits impaired channel function, suggesting a pathogenetic role for such mutation. The degree of functional impairment of channels incorporating KCNQ3 R330L subunits was larger than that of channels carrying another KCNQ3 mutation affecting the same codon but leading to a different amino acid substitution (p.R330C), previously identified in two families with typical BFNS. These data suggest that mutations in KCNQ3, similarly to KCNQ2, can be found in patients with more severe phenotypes including intellectual disability, and that the degree of the functional impairment caused by mutations at position 330 in KCNQ3 may contribute to clinical disease severity.
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http://dx.doi.org/10.1111/epi.12887DOI Listing
February 2015

Critical role of large-conductance calcium- and voltage-activated potassium channels in leptin-induced neuroprotection of N-methyl-d-aspartate-exposed cortical neurons.

Pharmacol Res 2014 Sep 26;87:80-6. Epub 2014 Jun 26.

Department of Medicine and Health Science, University of Molise, Campobasso, Italy; Department of Neuroscience, University of Naples "Federico II", Naples, Italy. Electronic address:

In the present study, the neuroprotective effects of the adipokine leptin, and the molecular mechanism involved, have been studied in rat and mice cortical neurons exposed to N-methyl-d-aspartate (NMDA) in vitro. In rat cortical neurons, leptin elicited neuroprotective effects against NMDA-induced cell death, which were concentration-dependent (10-100 ng/ml) and largest when the adipokine was preincubated for 2h before the neurotoxic stimulus. In both rat and mouse cortical neurons, leptin-induced neuroprotection was fully antagonized by paxilline (Pax, 0.01-1 μM) and iberiotoxin (Ibtx, 1-100 nM), with EC50s of 38 ± 10 nM and 5 ± 2 nM for Pax and Ibtx, respectively, close to those reported for Pax- and Ibtx-induced Ca(2+)- and voltage-activated K(+) channels (Slo1 BK channels) blockade; the BK channel opener NS1619 (1-30 μM) induced a concentration-dependent protection against NMDA-induced excitotoxicity. Moreover, cortical neurons from mice lacking one or both alleles coding for Slo1 BK channel pore-forming subunits were insensitive to leptin-induced neuroprotection. Finally, leptin exposure dose-dependently (10-100 ng/ml) increased intracellular Ca(2+) levels in rat cortical neurons. In conclusion, our results suggest that Slo1 BK channel activation following increases in intracellular Ca(2+) levels is a critical step for leptin-induced neuroprotection in NMDA-exposed cortical neurons in vitro, thus highlighting leptin-based intervention via BK channel activation as a potential strategy to counteract neurodegenerative diseases.
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http://dx.doi.org/10.1016/j.phrs.2014.06.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4134969PMC
September 2014

Novel KCNQ2 and KCNQ3 mutations in a large cohort of families with benign neonatal epilepsy: first evidence for an altered channel regulation by syntaxin-1A.

Hum Mutat 2014 Mar 13;35(3):356-67. Epub 2014 Jan 13.

Department of Medicine and Health Science, University of Molise, Campobasso, Italy.

Mutations in the KCNQ2 and KCNQ3 genes encoding for Kv 7.2 (KCNQ2; Q2) and Kv 7.3 (KCNQ3; Q3) voltage-dependent K(+) channel subunits, respectively, cause neonatal epilepsies with wide phenotypic heterogeneity. In addition to benign familial neonatal epilepsy (BFNE), KCNQ2 mutations have been recently found in families with one or more family members with a severe outcome, including drug-resistant seizures with psychomotor retardation, electroencephalogram (EEG) suppression-burst pattern (Ohtahara syndrome), and distinct neuroradiological features, a condition that was named "KCNQ2 encephalopathy." In the present article, we describe clinical, genetic, and functional data from 17 patients/families whose electroclinical presentation was consistent with the diagnosis of BFNE. Sixteen different heterozygous mutations were found in KCNQ2, including 10 substitutions, three insertions/deletions and three large deletions. One substitution was found in KCNQ3. Most of these mutations were novel, except for four KCNQ2 substitutions that were shown to be recurrent. Electrophysiological studies in mammalian cells revealed that homomeric or heteromeric KCNQ2 and/or KCNQ3 channels carrying mutant subunits with newly found substitutions displayed reduced current densities. In addition, we describe, for the first time, that some mutations impair channel regulation by syntaxin-1A, highlighting a novel pathogenetic mechanism for KCNQ2-related epilepsies.
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http://dx.doi.org/10.1002/humu.22500DOI Listing
March 2014

Subtype-selective activation of K(v)7 channels by AaTXKβ₂₋₆₄, a novel toxin variant from the Androctonus australis scorpion venom.

Mol Pharmacol 2013 Nov 9;84(5):763-73. Epub 2013 Sep 9.

Laboratoire des Venins et Molécules Thérapeutiques, Institut Pasteur de Tunis, Université Tunis-El Manar, Tunis-Belvédère, Tunisia (Z.L., M.E.A., R.B.); Division of Pharmacology, Department of Neuroscience, University of Naples Federico II, Naples, Italy (F.M., M.T.); Department of Chemical Sciences, University of Naples Federico II, Naples, Italy (A.P., A.A., G.M.); Department of Medicine and Health Science, University of Molise, Campobasso, Italy (M.T.); and Unidad de Biofísica, Consejo Superior de Investigaciones Cientificas, Universidad del Pais Vasco, Leioa, Spain (M.T.).

K(v)7.4 channel subunits are expressed in central auditory pathways and in inner ear sensory hair cells and skeletal and smooth muscle cells. Openers of K(v)7.4 channels have been suggested to improve hearing loss, systemic or pulmonary arterial hypertension, urinary incontinence, gastrointestinal and neuropsychiatric diseases, and skeletal muscle disorders. Scorpion venoms are a large source of peptides active on K⁺ channels. Therefore, we have optimized a combined purification/screening procedure to identify specific modulator(s) of K(v)7.4 channels from the venom of the North African scorpion Androctonus australis (Aa). We report the isolation and functional characterization of AaTXKβ₂₋₆₄, a novel variant of AaTXKβ₁₋₆₄, in a high-performance liquid chromatography fraction from Aa venom (named P8), which acts as the first peptide activator of K(v)7.4 channels. In particular, in both Xenopus oocytes and mammalian Chinese hamster ovary cells, AaTXKβ₂₋₆₄, but not AaTXKβ₁₋₆₄, hyperpolarized the threshold voltage of current activation and increased the maximal currents of heterologously expressed K(v)7.4 channels. AaTXKβ₂₋₆₄ also activated K(v)7.3, K(v)7.2/3, and K(v)7.5/3 channels, whereas homomeric K(v)1.1, K(v)7.1, and K(v)7.2 channels were unaffected. We anticipate that these results may prove useful in unraveling the novel biologic roles of AaTXKβ₂₋₆₄-sensitive K(v)7 channels and developing novel pharmacologic tools that allow subtype-selective targeting of K(v)7 channels.
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http://dx.doi.org/10.1124/mol.113.088971DOI Listing
November 2013

Genotype-phenotype correlations in neonatal epilepsies caused by mutations in the voltage sensor of K(v)7.2 potassium channel subunits.

Proc Natl Acad Sci U S A 2013 Mar 25;110(11):4386-91. Epub 2013 Feb 25.

Department of Neuroscience, University of Naples Federico II, 80131 Naples, Italy.

Mutations in the K(V)7.2 gene encoding for voltage-dependent K(+) channel subunits cause neonatal epilepsies with wide phenotypic heterogeneity. Two mutations affecting the same positively charged residue in the S4 domain of K(V)7.2 have been found in children affected with benign familial neonatal seizures (R213W mutation) or with neonatal epileptic encephalopathy with severe pharmacoresistant seizures and neurocognitive delay, suppression-burst pattern at EEG, and distinct neuroradiological features (R213Q mutation). To examine the molecular basis for this strikingly different phenotype, we studied the functional characteristics of mutant channels by using electrophysiological techniques, computational modeling, and homology modeling. Functional studies revealed that, in homomeric or heteromeric configuration with K(V)7.2 and/or K(V)7.3 subunits, both mutations markedly destabilized the open state, causing a dramatic decrease in channel voltage sensitivity. These functional changes were (i) more pronounced for channels incorporating R213Q- than R213W-carrying K(V)7.2 subunits; (ii) proportional to the number of mutant subunits incorporated; and (iii) fully restored by the neuronal K(v)7 activator retigabine. Homology modeling confirmed a critical role for the R213 residue in stabilizing the activated voltage sensor configuration. Modeling experiments in CA1 hippocampal pyramidal cells revealed that both mutations increased cell firing frequency, with the R213Q mutation prompting more dramatic functional changes compared with the R213W mutation. These results suggest that the clinical disease severity may be related to the extent of the mutation-induced functional K(+) channel impairment, and set the preclinical basis for the potential use of K(v)7 openers as a targeted anticonvulsant therapy to improve developmental outcome in neonates with K(V)7.2 encephalopathy.
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http://dx.doi.org/10.1073/pnas.1216867110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3600471PMC
March 2013