Neuronal K-Cl Cotransporter : Transcriptional Mechanisms of KCC2 Gene Regulation

K-Cl cotransporter 2 (KCC2) maintains a low intracellular Cl concentration required for fast hyperpolarizing responses of neurons to classical inhibitory neurotransmitters γ-aminobutyric acid (GABA) and glycine. Decreased Cl extrusion observed in genetically modified KCC2-deficient mice leads to depolarizing GABA responses, impaired brain inhibition, and as a consequence to epileptic seizures. Identification of mechanisms regulating activity of the SLC12A5 gene, which encodes the KCC2 cotransporter, in normal and pathological conditions is, thus, of extreme importance. Multiple reports have previously elucidated in details a spatio-temporal pattern of KCC2 expression. Among the characteristic features are an exclusive neuronal specificity, a dramatic upregulation during embryonic and early postnatal development, and a significant downregulation by neuronal trauma. Numerous studies confirmed these expressional features, however transcriptional mechanisms predetermining the SLC12A5 gene behaviour are still unknown. The aim of the presented thesis is to recognize such transcriptional mechanisms and, on their basis, to create a transcriptional model that would explain the established SLC12A5 gene behaviour. Up to recently, only one KCC2 transcript has been thought to exist. A particular novelty of the presented work is the identification of two SLC12A5 gene promoters (SLC12A5-1a and SLC12A5-1b) that produce at least two KCC2 isoforms (KCC2a and KCC2b) differing by their N-terminal parts. Even though a functional 86Rb+ assay reveals no significant difference between transport activities of the isoforms, consensus sites for several protein kinases, found in KCC2a but not in KCC2b, imply a distinct kinetic regulation. As a logical continuation, the current work presents a detailed analysis of the KCC2a and KCC2b expression patterns. This analysis shows an exclusively neuron-specific pattern and similar expression levels for both isoforms during embryonic and neonatal development in rodents. During subsequent postnatal development, the KCC2b expression dramatically increases, while KCC2a expression, depending on central nervous system (CNS) area, either remains at the same level or moderately decreases. In an attempt to explain both the neuronal specificity and the distinct expressional kinetics of the KCC2a and KCC2b isoforms during postnatal development, the corresponding SLC12A5-1a and SLC12A5-1b promoters have been subjected to a comprehensive bioinformatical analysis. Binding sites of several transcription factors (TFs), conserved in the mammalian SLC12A5 gene orthologs, have been identified that might shed light on the observed behaviour of the SLC12A5 gene. Possible roles of these TFs in the regulating of the SLC12A5 gene expression have been elucidated in subsequent experiments and are discussed in the current thesis.Oikea ionikonsentraatio on välttämätön elimistön normaalissa toiminnassa. Monia ionien kuljettajia, kanavia ja pumppuja tarvitaan tähän. Häiriöt näiden proteiinien toiminnassa aiheuttavat erilaisia sairauksia. Tässä väitöskirjatyössä keskitytään KCC2 proteiiniin, joka on tarpeen kaliumin ja kloridin liikenteessä solukalvon läpi. KCC2 proteiinin väheneminen hiirillä johtaa hermosolujen hyperaktivaatioon, mikä puolestaan voi aiheuttaa epileptisiä kohtauksia. Hiiret, joilta KCC2 puuttuu, kuolevat pian syntymän jälkeen, koska ne eivät voi hengittää. Kyvyttömyys hengittää todennäköisimmin liittyy siihen, että neuronien hyperaktivaatio rikkoo hengitysteiden rytmiä ylläpitävän mekanismin. Toisin kuin monet muut elimistön proteiinit, KCC2 kuljetusproteiini ilmenee ainoastaan keskushermoston hermosoluissa. Tässä tutkimuksessa tutkittiin mekanismeja, jotka estävät KCC2 proteiinin tuotanto muissa soluissa kuin hermosoluissa. Kuten edellä mainittiin, tuotetun KCC2 proteiinin väheneminen aiheuttaa vakavia seurauksia. Tutkimuksessa myös pyrittiin vastaamaan kysymykseen, mitkä mekanismit ylläpitävät tämän kuljettajan tarvittavaa tasoa soluissa

A noninvasive optical approach for assessing chloride extrusion activity of the K-Cl cotransporter KCC2 in neuronal cells

Background: Cation-chloride cotransporters (CCCs) are indispensable for maintaining chloride homeostasis in multiple cell types, but K-Cl cotransporter KCC2 is the only CCC member with an exclusively neuronal expression in mammals. KCC2 is critical for rendering fast hyperpolarizing responses of ionotropic.-aminobutyric acid and glycine receptors in adult neurons, for neuronal migration in the developing central nervous system, and for the formation and maintenance of small dendritic protrusions-dendritic spines. Deficit in KCC2 expression and/or activity is associated with epilepsy and neuropathic pain, and effective strategies are required to search for novel drugs augmenting KCC2 function. Results: We revised current methods to develop a noninvasive optical approach for assessing KCC2 transport activity using a previously characterized genetically encoded chloride sensor. Our protocol directly assesses dynamics of KCC2-mediated chloride efflux and allows measuring genuine KCC2 activity with good spatial and temporal resolution. As a proof of concept, we used this approach to compare transport activities of the two known KCC2 splice isoforms, KCC2a and KCC2b, in mouse neuronal Neuro-2a cells. Conclusions: Our noninvasive optical protocol proved to be efficient for assessment of furosemide-sensitive chloride fluxes. Transport activities of the N-terminal splice isoforms KCC2a and KCC2b obtained by the novel approach matched to those reported previously using standard methods for measuring chloride fluxes.Peer reviewe

Origin and Loss of Nested LRRTM/alpha-Catenin Genes during Vertebrate Evolution

Peer reviewe

Кто глаголет устами младенца? Дети и религиозное насилие во Франции. Рец. на кн.: Crouzet D. Les enfants bourreaux au temps des guerres de Religion. — Paris : Albin Michel, 2020. — 336 p.

This essay contains reflections on a new book by renowned historian Denis Crouzet on children’s violence, and, more broadly, on the image of children during the French Wars of Religion. In the book under review, the novelty lies in the fact that the images of ‘innocent infants’ make part of a separate plot. Just as novel are Denis Crouzet’s reflections on the ‘sources of inspiration’ of the young French persecutors of heretics. The author indicates the anthropological correspondences inherent in the culture of both Italian and French cities, such as the carnivalesque inversion of the ‘world inside out’ and the social function of youth associations taking part in the ‘charivari’ rites. Denis Crouzet pays attention to sources that are novel to him, like children’s Christmas chants, mystery plays, and ‘miracles’. While impersonating the Innocents persecuted by Herod but also angels carrying retaliation to this villain, urban children learnt what and how to do in the face of a carnival challenge.The ways to leave the eschatological activism are of particular interest. After 1572, the gangs of executioners-children left the scene. Only the murder of the Guises on Christmas Day, 1588, threw crowds of children into the streets of Paris. Now they were described differently, however, — as a disciplined mass, occupied not with outrages but with prayers. The author speaks of ‘Catholic consciousness’, but that was already a different reformed Catholicism, departing further and further from the old ‘corporate Catholicism’. The religious political activity of children would become a thing of the past, however. The image of an innocent child would once more be in demand only after the Revolution, when, this time in a desacralised context, children became the embodiment of the French nation.Данное эссе представляет собой размышления над новой книгой известного французского историка Дени Крузе, посвященной теме детского насилия, и шире — образу детей во время Религиозных войн во Франции. Новизна и ценность рассматриваемой книги заключается в выделении образов «невинных младенцев» в отдельный сюжет. Новыми представляются и размышления автора об «истоках вдохновения» юных французских гонителей гугенотов. Д. Крузе указывает на антропологические соответствия, присущие культуре как итальянских, так и французских городов. Здесь и карнавальная инверсия «мира наизнанку», и социальная функция объединений молодежи, участвующих в обрядах шаривари. Исследователь обращается к новым источникам: песнопениям, исполняемым детьми на Рождество, к мистериям и мираклям. Играя как невинных младенцев, страдающих от Ирода, так и ангелов, карающих злодея, городские дети учились тому, что и как делать в условиях карнавального вызова.Интерес представляет процесс выхода из эсхатологического активизма. После 1572 г. банды детей-палачей сходят со сцены. Лишь убийство Гизов на Рождество 1588 г. выплеснуло на улицы Парижа толпы детей. Но теперь они описывались иначе — как дисциплинированная масса, занятая не погромами, но молитвами. Крузе говорит о «католическом сознании», но это был уже иной католицизм, реформированный, все далее отходящий от старого «корпоративного католицизма». Религиозная политическая активность детей уйдет в прошлое. Вновь образ невинного ребенка будет востребован уже после Революции, когда в десакрализованном контексте дети станут воплощением французской нации

Проекты реформ Галликанской церкви и «повсе­дневного благочестия» накануне Религиозных войн

During the so-called ‘Gallican crisis’ between 1551 and 1552, Pope Julius III accused the French king of preparing an ecclesiastical schism, while the possibility of establishing a French patriarchate was discussed in the royal council. Before long, however, the conflict gave way to a close alliance between the Pope and King Henry II. Was the ‘Gallican crisis’ just a tool of political pressure on Julius III? To what extent were the plans of the king and his entourage to reform the Gallican Church serious? The lack of sources can be filled, at least in part, by turning to the work by Raoul Spifame, a lawyer of the Paris Parliament, titled Dicaearchiae Henrici Regis Christianissimi Progymnasmata (1556). In its essence, it is a collection of rhetorical exercises in the field of jurisprudence written in the form of royal decrees designed to reform everything in the kingdom. Surprisingly, some of these fictional measures later would be actually implemented. The reason for the author’s ‘clairvoyance’ lies in his contacts with the secretaries of state who were then preparing large-scale reforms, which would eventually be cancelled due to the unexpected death of the king and the outbreak of the Wars of Religion. A considerable part of the decrees is devoted to plans of the reform of the Gallican Church: from the elevation of the Bishopric of Paris to the rank of an archdiocese to tightening control over the morals of prelates. This article pays special attention to how the Dicaearchiae regulated the elements of ‘everyday piety’ — the rituals of blessing of the bridal chamber, purification after childbirth, and belief in the existence of limb. A limitation of ‘luxury’ was also to be introduced: refusal of precious ecclesiastical ornaments, redundant bells, and a reduction in the number of holidays associated with the veneration of saints. Without abandoning the cult of saints, Spifame undertakes a reform of the ecclesiastical calendar and creates a sort of national martyrology of warriors who died for their homeland and ‘are venerated as saints without a canonisation’.Во время так называемого «галликанского кризиса» 1551–1552 гг. папа Юлий III обвинял французского короля в подготовке схизмы, а в Королевском совете обсуждалась возможность учреждения патриаршества во Франции. Но вскоре на смену конфликту пришел тесный союз папы и Генриха II. Был ли «галликанский кризис» лишь средством политического давления на Юлия III? Насколько серьезны были планы короля и его окружения по реформированию Галликанской церкви? Недостаток источников, освещавших этот вопрос, можно в определенной мере восполнить за счет анализа сочинения адвоката Парижского парламента Рауля Спифама «Dicaearchiae Henrici Regis Christianissimi Progymnasmata» (1556). Это был сборник риторических упражнений в области юриспруденции, написанный в форме королевских постановлений о реформировании всех дел в королевстве. Удивительным образом часть этих вымышленных проектов реализовалась в будущем. Причина «ясновидения» автора крылась в его связях с государственными секретарями, готовившими масштабные преобразования, которые остались нереализованными из-за неожиданной гибели короля и начавшихся Религиозных войн. Немалая часть постановлений посвящена планам реформы Галликанской церкви: от возведения епископства Парижского в архиепископский ранг до ужесточения контроля над нравами прелатов. В данной статье особое внимание уделяется тому, как в «Dicaearchiae» регламентировалась форма ритуалов «повседневного благочестия»: благословения ложа новобрачных, очищения женщины после родов перед посещением церкви, представлений о лимбе. Предусматривалось ограничение «роскоши» — отказ от драгоценной церковной утвари, излишних колоколов, сокращение числа праздничных дней, связанных с почитанием святых. Не отказываясь от культа святых, Спифам предпринимает реформу церковного календаря и создает особый национальный мартиролог воинов, погибших за родину и «почитаемых как святые без всякой канонизации»

Город чист, безопасен и хорошо управляем: урбанизм, утопизм и реализм в восприятии Парижа времен Генриха II

Sixteenth-century French kings paid much attention to decorating cities and especially Paris. Henry II (1546–1559) did the most for the capital, but large-scale urban projects that reflect the image of an ideal city have not survived since his time. The image of an ideal city in the era of Henry II can be restored to some extent by analysing Dicaearchiae Henrici Regis Christianissimi Progymnasmata (1556), a work of the avocat de Parlement de Paris Raoul Spifamе.It was a collection of rhetorical exercises in the field of jurisprudence written in the form of royal arrêts and designed to reform all matters in the kingdom. This book has a controversial reputation. Some mistakenly believed that these were authentic laws of the king. Others considered it a sharp satire and even delirium of a madman. Still others were astonished at Spifame’s clairvoyance, noting that many of his imaginary projects were subsequently implemented. A considerable part of the arrêts is devoted to the problems of the city, i.e. its safety, cleanliness, maintaining the piety of citizens, helping the poor, orphans, and the diseased, reorganising the urban space. Spifamе planned a large-scale construction of a new large bridge across the Seine, a consolidation of deserted islands into one and its development, the creation of new ports, markets, and embankments. A significant part of these projects was implemented in the future. This “clairvoyance” of the author was due to his ties with secrétaires d’État preparing for large-scale transformations of the mid-sixteenth century which were never realised due to the unexpected death of the king and the outbreak of the Religious Wars.Французские короли XVI в. много внимания уделяли украшению городов и особенно Парижа. Больше всего для столицы сделал Генрих II (1546–1559), но от его времени не сохранилось масштабных урбанистических проектов, рисующих образ идеального города. Представления об идеальном городе в эпоху Генриха II можно в определенной мере восполнить за счет анализа сочинения адвоката Парижского парламента Рауля Спифама «Dicaearchiae Henrici Regis Christianissimi Progymnasmata» (1556). По сути, это был сборник риторических упражнений в области юриспруденции, написанный в форме королевских постановлений, призванных реформировать все дела в королевстве. У этой книги неоднозначная репутация. Одни принимали ее постановления за аутентичные законы короля. Другие считали ее острой сатирой и даже бредом сумасшедшего. Третьи удивлялись дару пророчества Спифама, отмечая, что многие из его фантастических проектов были впоследствии реализованы. Немалая часть постановлений посвящена проблемам города — его безопасности, чистоте, поддержанию благочестия горожан, помощи бедным, сиротам и больным, реорганизации городского пространства. Спифам планировал масштабное строительство нового моста через Сену, застройку пустующих островов, создание новых портов, рынков и набережных. Значительная часть этих проектов была реализована в будущем. Причина такого «ясновидения» автора крылась в его связях с государственными секретарями, готовившими масштабные преобразования середины XVI в., не реализованные из-за неожиданной гибели короля и начавшихся Религиозных войн

NKCC1, an Elusive Molecular Target in Brain Development: Making Sense of the Existing Data

Ionotropic GABA transmission is mediated by anion (mainly Cl−)-permeable GABAA receptors (GABAARs). In immature neurons, GABA exerts depolarizing and sometimes functionally excitatory actions, based on active uptake of Cl− by the Na-K-2Cl cotransporter NKCC1. While functional evidence firmly shows NKCC1-mediated ion transport in immature and diseased neurons, molecular detection of NKCC1 in the brain has turned out to be extremely difficult. In this review, we describe the highly inconsistent data that are available on the cell type-specific expression patterns of the NKCC1 mRNA and protein in the CNS. We discuss the major technical caveats, including a lack of knock-out-controlled immunohistochemistry in the forebrain, possible effects of alternative splicing on the binding of antibodies and RNA probes, and the wide expression of NKCC1 in different cell types, which make whole-tissue analyses of NKCC1 useless for studying its neuronal expression. We also review novel single-cell RNAseq data showing that most of the NKCC1 in the adult CNS may, in fact, be expressed in non-neuronal cells, especially in glia. As future directions, we suggest single-cell NKCC1 mRNA and protein analyses and the use of genetically tagged endogenous proteins or systematically designed novel antibodies, together with proper knock-out controls, for the visualization of endogenous NKCC1 in distinct brain cell types and their subcellular compartments

Loss of non-canonical KCC2 functions promotes developmental apoptosis of cortical projection neurons

KCC2, encoded in humans by the SLC12A5 gene, is a multifunctional neuron-specific protein initially identified as the chloride (Cl-) extruder critical for hyperpolarizing GABA(A) receptor currents. Independently of its canonical function as a K-Cl cotransporter, KCC2 regulates the actin cytoskeleton via molecular interactions mediated through its large intracellular C-terminal domain (CTD). Contrary to the common assumption that embryonic neocortical projection neurons express KCC2 at non-significant levels, here we show that loss of KCC2 enhances apoptosis of late-born upper-layer cortical projection neurons in the embryonic brain. In utero electroporation of plasmids encoding truncated, transport-dead KCC2 constructs retaining the CTD was as efficient as of that encoding full-length KCC2 in preventing elimination of migrating projection neurons upon conditional deletion of KCC2. This was in contrast to the effect of a full-length KCC2 construct bearing a CTD missense mutation (KCC2(R952H)), which disrupts cytoskeletal interactions and has been found in patients with neurological and psychiatric disorders, notably seizures and epilepsy. Together, our findings indicate ion transport-independent, CTD-mediated regulation of developmental apoptosis by KCC2 in migrating cortical projection neurons.Peer reviewe

The Multifaceted Roles of KCC2 in Cortical Development

KCC2, best known as the neuron-specific chloride-extruder that sets the strength and polarity of GABAergic currents during neuronal maturation, isa multifunctional molecule that can regulate cytoskeletal dynamics via its C-terminal domain (CTD). We describe the molecular and cellular mechanisms involved in the multiple functions of KCC2 and its splice variants, ranging from developmental apoptosis and the control of early network events to the formation and plasticity of cortical dendritic spines. The versatility of KCC2 actions at the cellular and subcellular levels is also evident in mature neurons during plasticity, disease, and aging. Thus, KCC2 has emerged as one of the most important molecules that shape the overall neuronal phenotype.Peer reviewe

Avant-propos

En 2003, le Centre d’études du monde russe, soviétique et post-soviétique de l’EHESS (aujourd’hui Centre d’études des mondes russe, caucasien et centre-européen, ou CERCEC) à Paris a proposé aux jeunes chercheurs d’Europe, du Japon, des États-Unis un séminaire doctoral autour du thème : « Naissance d’une monarchie nationale et formation d’un État russe (milieu du xive-milieu du xvie siècle) dans le contexte européen ». Organisé conjointement par le Centre et par l’Institut d’histoire universe..