Microglia-Mediated Inflammation and Neural Stem Cell Differentiation in Alzheimer’s Disease: Possible Therapeutic Role of KV1.3 Channel Blockade

Increase of deposits of amyloid beta peptides in the extracellular matrix is landmark during Alzheimer's Disease (AD) due to the imbalance in the production vs. clearance. This accumulation of amyloid beta deposits triggers microglial activation. Microglia plays a dual role in AD, a protective role by clearing the deposits of amyloid beta peptides increasing the phagocytic response (CD163, IGF-1 or BDNF) and a cytotoxic role, releasing free radicals (ROS or NO) and proinflammatory cytokines (TNF-alpha, IL-1beta) in response to reactive gliosis activated by the amyloid beta aggregates. Microglia activation correlated with an increase KV1.3 channels expression, protein levels and current density. Several studies highlight the importance of KV1.3 in the activation of inflammatory response and inhibition of neural progenitor cell proliferation and neuronal differentiation. However, little is known about the pathways of this activation in neural stem cells differentiation and proliferation and the role in amyloid beta accumulation. In recent studies using in vitro cells derived from mice models, it has been demonstrated that KV1.3 blockers inhibit microglia-mediated neurotoxicity in culture reducing the expression and production of the pro-inflammatory cytokines IL-1beta and TNF-alpha through the NF-kB and p38MAPK pathway. Overall, we conclude that KV1.3 blockers change the course of AD development, reducing microglial cytotoxic activation and increasing neural stem cell differentiation. However, further investigations are needed to establish the specific pathway and to validate the use of this blocker as therapeutic treatment in Alzheimer patients.This work was supported by a grant from the MICINN (PID2020-118814RB-I00), the Government of the Autonomous Community of the Basque Country (IT1165-19 and KK-2020/00110), and the Spanish Ministry of Science and Innovation (RTI2018-097839-B-100 to AV)

Gure bihotzeko kalmodulina

Calcium is a universal signaling messenger that participates in essential processes such as apoptosis, cell proliferation and muscle contraction. Many of the proteins involved in these processes must interact with a calcium sensor to respond to the changes of concentration of this ion. The best-studied sensor is Calmodulin (CaM). It regulates the activity of hundreds of proteins through its N- and C- lobes, where two EF-hands are located to bind up to four calcium ions. Although the role of CaM in cell signaling is widespread, its mutations are especially recognized through its effects on cardiac function. In fact, disease-causing mutations in any of the three genes that encode the same CaM proteins cause severe cardiac dysfunction, indicating their importance in regulating excitability. Therefore, knowing the mechanisms involved in these diseases can allow a rational approach to clinical manifestations and contribute to the development of therapeutic strategies.; Kaltzioa seinalizazio unibertsaleko mezulari bat da, funtsezko prozesuetan parte hartzen duena, hala nola apoptosian, zelulen proliferazioan eta muskuluen uzkurduran. Prozesu horietan parte hartzen duten proteina askok ioi honen kontzentrazioari erantzuteko kaltzio sentsore batekin, kalmodulinarekin (CaM), elkarreragin behar dute. Horrek ehunka proteinaren aktibitatea erregulatzen du bere N- eta C-lobuluen bidez, non EF-eskuak aurkezten dituen Ca2+-ari lotzeko. Zelulen seinalizazioan CaM-k duen papera hedatuta dagoen arren, haren mutazioek bereziki bihotzari eragiten diote. Izan ere, CaM proteina berdinak kodetzen dituzten hiru geneetako edozeinetan gaixotasunak eragiten dituzten mutazioek bihotz-disfuntzio larriak eragiten dituzte, eta horrek kitzikagarritasunaren erregulazioan duen garrantzia adierazten du. Beraz, esku hartzen duten mekanismoen ezagutzak aukera eman dezake adierazpen klinikoei modu kritikoan heltzeko eta kalmodulinopatietarako estrategia terapeutikoak garatzen laguntzeko

The Crossroad of Ion Channels and Calmodulin in Disease

Calmodulin (CaM) is the principal Ca2+ sensor in eukaryotic cells, orchestrating the activity of hundreds of proteins. Disease causing mutations at any of the three genes that encode identical CaM proteins lead to major cardiac dysfunction, revealing the importance in the regulation of excitability. In turn, some mutations at the CaM binding site of ion channels cause similar diseases. Here we provide a summary of the two sides of the partnership between CaM and ion channels, describing the diversity of consequences of mutations at the complementary CaM binding domains.The Department of Industry, Tourism and Trade of the Government of the Autonomous Community of the Basque Country (Elkartek 2017 bG17 kk-2017/000843M50.17.EK.C6) and the Spanish Ministry of Economy, Industry and Competitiveness (BFU2015-66910 and RTI2018-097839) provided financial support for this work. E.N. is supported by a predoctoral grant of the Basque Government

Proteinen tolestura tunel erribosomikoan

Proteins are synthesised as linear polymers and must fold into their native three-dimensional structure to perform various functions in the cell. Understanding protein folding is crucial because protein misfolding is at the origin of several neurodegenerative diseases. Protein folding can start cotranslationally, i.e. when the emerging peptide is still asso-ciated with the ribosome. Indeed, it has been shown that more than one third of the cell’s proteins fold in the limited space of the ribosome tunnel. Increasing evidence suggests that the ribosome plays a critical role in protein folding. The ribosome can facilitate protein compaction, cause the creation of non-visible media in solution or delay the onset of folding. However, the study of cotranslational folding presents serious difficulties, mainly due to the limitations of the different current techniques. Hence, most studies on protein folding are based on proteins in solution, which are carried out by unfolding and refolding the protein, without taking into account the role of the ribosome in this process. In this article, we summarised the techniques developed in recent years for the study of cotranslational protein folding.; Proteinak polimero lineal gisa sintetizatzen dira eta beren jatorrizko egitura tridimentsionalean tolestu behar dira zelulan hainbat funtzio betetzeko. Proteinen tolespena ulertzea funtsezkoa da, tolespen okerrak hainbat gaixotasun neuro-degeneratiboren jatorria direlako. Proteinen tolespena modu koitzultzailean has daiteke, hau da, sortzen ari den peptidoa erribosomari lotuta dagoenean oraindik. Izan ere, zelularen proteinen heren bat baino gehiago erribosomaren tunelaren espazio mugatuan tolesten direla frogatu da, hau da, erribosomaren gainazalarekiko interakzioek modulatuta eta erribosoma-tunelaren beraren mugen pean. Gero eta ebidentzia gehiagok iradokitzen dute erribosomak funtsezko zeregina duela proteinen tolespenean. Erribosomak proteina trinkotzea erraztu dezake, soluzioan ikusten ez diren bitartekoak sortzea eragin dezake edo tolestearen hasiera atzeratu dezake. Hala ere, proteinen koitzulpenezko tolesdura aztertzeak zailtasun handiak ditu, batik bat, egungo teknikek dituzten mugengatik. Hori dela eta, proteinen tolesteari buruzko ikerketa gehienak soluzioan dauden proteinetan oinarritzen dira, proteina tolestuz eta destolestuz egiten direnak, prozesu horretan erribosomak duen rola kontuan hartu gabe. Artikulu honetan, azken urteotan proteinen koitzulpenezko tolestura ikertzeko garatu diren tekniken laburpena egin da

KV7.2 kanala: estruktura, erregulazioa eta kitzikagarritasun neuronalean duen ekintza

Potasio-kanalak ia zelula guztien mintzean agertzen dira eta funtzio biologiko garrantzitsuak betetzen dituzte; besteak beste, korronte elektrikoak kontrolatzen dituzte zelula kitzikagarrietan. KV7 kanalen familia 5 kidez osatuta dago (KV7.1-KV7.5), eta horiek kodetzen dituzten geneak patologia esanguratsuekin erlazionatzen dira. KV7 kanalen estrukturak zelula-mintzean txertaturiko 6 segmentuz osaturiko ohiko estruktura partekatzen du; N- eta C-muturrak zelula barnekoak dira. Neuronetan, KV7.2 eta KV7.3 kanalak agertzen dira batik bat; M-korrontea sortuz, neuronen kitzikagarritasuna kontrolatzen duena. M-korrontearen erregulazioa konplexua da seinaleztapen-bidezidor desberdinen bidez erregula baitaiteke. Gq/11 proteinari akoplaturiko hartzaileen bidez erregulatzen da eta seinaleztapen-bidezidorra desberdina da aktibatutako hartzailearen arabera. Horrela, azetilkolinaren M1 hartzaile muskarinikoak KV7.2-aren korrontea inhibituko du PIP2-aren agorpenaren ondorioz. Bradikininaren hartzaileak, ordea, IP3-ak eragindako kaltzio-kontzentrazioaren igoeraren bidez inhibituko du. Mekanismo horietan, hainbat proteinak hartzen dute parte, hala nola kalmodulinak, proteina kinasek eta ainguratze-proteinek. Berrikuspen honetan, KV7.2 kanalari erreparatuko diogu, hainbat gaixotasunen partaide izateagatik eta haren erregulazio konplexuagatik, ikuspuntu farmakologiko batetik itu interesgarria izan baitaiteke.; Potassium channels are present in almost all cell membranes and perform important biological functions, including electrical currents control in excitable cells. The KV7 channels familiy consists of 5 members (KV7.1-KV7.5) and the genes that encode them are related to significant pathologies. The structure of KV7 channels shares the usual six transmem-brane segment structure, with intracellular N- and C-termini. In neurons, KV7.2 and KV7.3 are the main channels, which generate the M-current that controls neuronal excitability. The M-current regulation is complex as it can be regulated by different signalling pathways. It is regulated by Gq/11-coupled receptors, and the signaling pathway depends on the activated receptor. Thus, the M1 muscarinic acetylcholine receptor inhibits KV7.2 current by PIP2 depletion. While the bradykinin receptor inhibits it through the calcium concentration increase driven by IP3. Among these mechanisms several proteins are involved, such as calmodulin, protein kinases and anchor proteins. In this review we will focus on KV7.2 channel, as it is involved in several diseases and for its complex regulation it can be an interesting target from a pharmacological point of view

Redox regulation of KV7 channels through EF3 hand of calmodulin

Neuronal KV7 channels, important regulators of cell excitability, are among the most sensitive proteins to reactive oxygen species. The S2S3 linker of the voltage sensor was reported as a site-mediating redox modulation of the channels. Recent structural insights reveal potential interactions between this linker and the Ca2+-binding loop of the third EF-hand of calmodulin (CaM), which embraces an antiparallel fork formed by the C-terminal helices A and B, constituting the calcium responsive domain (CRD). We found that precluding Ca2+ binding to the EF3 hand, but not to EF1, EF2, or EF4 hands, abolishes oxidation-induced enhancement of KV7.4 currents. Monitoring FRET (Fluorescence Resonance Energy Transfer) between helices A and B using purified CRDs tagged with fluorescent proteins, we observed that S2S3 peptides cause a reversal of the signal in the presence of Ca2+ but have no effect in the absence of this cation or if the peptide is oxidized. The capacity of loading EF3 with Ca2+ is essential for this reversal of the FRET signal, whereas the consequences of obliterating Ca2+ binding to EF1, EF2, or EF4 are negligible. Furthermore, we show that EF3 is critical for translating Ca2+ signals to reorient the AB fork. Our data are consistent with the proposal that oxidation of cysteine residues in the S2S3 loop relieves KV7 channels from a constitutive inhibition imposed by interactions between the EF3 hand of CaM which is crucial for this signaling.Ministerio de Ciencia e Innovacion PID2021-128286NB-100Wellcome Trust 212302/Z/18/ZMedical Research Centre MR/P015727/1Eusko Jaurlaritza IT1707-22 Ekonomiaren Garapen eta Lehiakortasun Saila, Eusko Jaurlaritza BG2019Ministerio de Ciencia e Innovacion RTI2018-097839-B-100Ministerio de Ciencia e Innovacion RTI2018-101269-B-I00Eusko Jaurlaritza IT1165-19 Ekonomiaren Garapen eta Lehiakortasun Saila,Eusko Jaurlaritza KK-2020/00110Eusko Jaurlaritza PRE_2018-2_0082Eusko Jaurlaritza POS_2021_1_0017Eusko Jaurlaritza PRE_2018-2_012

An Epilepsy-Causing Mutation Leads to Co-Translational Misfolding of the Kv7.2 Channel

BACKGROUND: The amino acid sequence of proteins generally carries all the necessary information for acquisition of native conformations, but the vectorial nature of translation can additionally determine the folding outcome. Such consideration is particularly relevant in human diseases associated to inherited mutations leading to structural instability, aggregation, and degradation. Mutations in the KCNQ2 gene associated with human epilepsy have been suggested to cause misfolding of the encoded Kv7.2 channel. Although the effect on folding of mutations in some domains has been studied, little is known of the way pathogenic variants located in the calcium responsive domain (CRD) affect folding. Here, we explore how a Kv7.2 mutation (W344R) located in helix A of the CRD and associated with hereditary epilepsy interferes with channel function. RESULTS: We report that the epilepsy W344R mutation within the IQ motif of CRD decreases channel function, but contrary to other mutations at this site, it does not impair the interaction with Calmodulin (CaM) in vitro, as monitored by multiple in vitro binding assays. We find negligible impact of the mutation on the structure of the complex by molecular dynamic computations. In silico studies revealed two orientations of the side chain, which are differentially populated by WT and W344R variants. Binding to CaM is impaired when the mutated protein is produced in cellulo but not in vitro, suggesting that this mutation impedes proper folding during translation within the cell by forcing the nascent chain to follow a folding route that leads to a non-native configuration, and thereby generating non-functional ion channels that fail to traffic to proper neuronal compartments. CONCLUSIONS: Our data suggest that the key pathogenic mechanism of Kv7.2 W344R mutation involves the failure to adopt a configuration that can be recognized by CaM in vivo but not in vitroThe Government of the Autonomous Community of the Basque Country (IT1165-19 and KK-2020/00110) and the Spanish Ministry of Science and Innovation (RTI2018-097839-B-100 to A.V. and FIS2016-76617-P to A.B.) and FEDER funds and the US National Institute of Neurological Disorders (NINDS) and Stroke Research Project Grant (R01NS083402 to H.J.C.) provided financial support for this work. E.N. and A.M-M. are supported by predoctoral contracts from the Basque Government administered by University of the Basque Country. C.M. was supported by the Basque Government through a Basque Excellence Research Centre (BERC) grant administered by Fundación Biofisika Bizkaia (FBB). J.U. was partially supported by BERC funds. O.R.B. was supported by the Basque Government through a BERC grant administered by Donostia International Physics Center. J.Z. and H.J.C. was supported by the NINDS Research Project Grant #R01NS083402 (PI: H.J.C.)

Regulación alfa1-adrenérgica de la corriente IKr cardiaca

149 p. : il., graf.Las distintas demandas cardiovasculares del corazón requieren respuestas dinámicas. El ritmo cardiaco y la contractilidad son regulados por complejos hormonales y cascadas de señalización autónomas implicando receptores acoplados a proteínas G. En particular la estimulación de ¿1-AR en cardiomiocitos provoca un alargamiento en la repolarización, un aumento moderado del inotropismo y un descenso del automatismo de las células de Purkinje [Kurz et ál., 1991], particularmente durante periodos de estrés extremo o una estimulación autónoma. Bajo condiciones patofisiológicas, resultados mal-adaptativos pueden inducir eventos severos cardiovasculares. Así, las arritmias ventriculares son a menudo precipitadas por estrés físico o emocional en particular en enfermos coronarios o con LQTS [Lown, 1987; Kamarck & Jennings, 1991; Napolitano et ál., 1994; Priori et ál., 1997]. Los resultados obtenidos en este trabajo, indican que la regulación de la corriente hERG por el receptor ¿1A-AR está mediada por la interacción entre PIP2 y el canal, con lo cual la disminución de la concentración de PIP2 desplaza el Vh de activación del canal hERG. Además, el Vh de activación de la corriente hERG está desplazado también, aunque en menor medida por la activación de PKC [figura 73]. Este desplazamiento del Vh de activación hacia potenciales más positivos, reduce la disponibilidad de la corriente a potenciales fisiológicos, provocando el alargamiento de la duración del potencial de acción cardiaco

Regulación alfa1-adrenérgica de la corriente IKr cardiaca

149 p. : il., graf.Las distintas demandas cardiovasculares del corazón requieren respuestas dinámicas. El ritmo cardiaco y la contractilidad son regulados por complejos hormonales y cascadas de señalización autónomas implicando receptores acoplados a proteínas G. En particular la estimulación de ¿1-AR en cardiomiocitos provoca un alargamiento en la repolarización, un aumento moderado del inotropismo y un descenso del automatismo de las células de Purkinje [Kurz et ál., 1991], particularmente durante periodos de estrés extremo o una estimulación autónoma. Bajo condiciones patofisiológicas, resultados mal-adaptativos pueden inducir eventos severos cardiovasculares. Así, las arritmias ventriculares son a menudo precipitadas por estrés físico o emocional en particular en enfermos coronarios o con LQTS [Lown, 1987; Kamarck & Jennings, 1991; Napolitano et ál., 1994; Priori et ál., 1997]. Los resultados obtenidos en este trabajo, indican que la regulación de la corriente hERG por el receptor ¿1A-AR está mediada por la interacción entre PIP2 y el canal, con lo cual la disminución de la concentración de PIP2 desplaza el Vh de activación del canal hERG. Además, el Vh de activación de la corriente hERG está desplazado también, aunque en menor medida por la activación de PKC [figura 73]. Este desplazamiento del Vh de activación hacia potenciales más positivos, reduce la disponibilidad de la corriente a potenciales fisiológicos, provocando el alargamiento de la duración del potencial de acción cardiaco