Novel regulation of AMPA receptor function by interacting protein CPT1C

[eng] AMPA receptors (AMPARs) are responsible for the 90% of synaptic transmission and are involved in plasticity, developmental and neurological processes. Their function depends on the proteins interacting with the AMPAR complex, which determine their specific gating and trafficking properties and hence their specific roles. In addition to well-known interacting proteins of AMPARs, recent studies have described the CPT1C as an associated partner in the outer core of the AMPAR complex in the hippocampus, cortex and cerebellum. CPT1C is a neuron specific homologue of the carnitine acyltransferase family of enzymes, which are involved in fatty acid oxidation at the mitochondria. Contrary to the rest of the CPT1 family, CPT1C localizes at the endoplasmic reticulum and apparently is not related with the functions that other CPT1s carry out. To elucidate the physiological role of CPT1C isoform different studies have been performed, showing that CPT1C is involved in energy homeostasis, control of body weight and motor function as well as behavioral learning mechanisms. Of note, CPT1C KO animals display impairments in spatial learning along with immature dendritic spines. Despite all these studies with CPT1C, including its interaction with AMPARs, it is unknown the CPT1C physiological relevance and role, particularly in the regulation of the AMPAR function and its implication in neurological diseases. In this thesis we have described a novel role of CPT1C in AMPA receptor function regulation. More specifically, the results show that glutamate-evoked currents of the recombinant GluA1 receptors are increased when CPT1C is present and this effect is specific of the CPT1 isoform CPT1C, since CPT1A does not share the same pattern. The ER location of CPT1C seems to be crucial to modulate AMPAR surface expression since mislocalization of CPT1C avoids its AMPAR modulation. Co-localization studies confirmed that GluA1-CPT1C interaction happens at ER level but not at the cell surface. On the other hand, no changes in current density have been found in cells expressing GluA2 subunit along with CPT1C indicating AMPA subunit specificity. Additionally, electrophysiological experiments have determined that GluA1 channel properties are no altered, thus indicating that the increased current is probably due to a rise in AMPAR number at the cell surface. Indeed, this hypothesis is corroborated by the findings from the immunofluorescence experiments, where surface expression of GluA1 is increased in the presence of CPT1C in heterologous systems and cortical neurons. In agreement with a putative role of CPT1C in determining the AMPAR content at synapse level, we have demonstrated that synaptic transmission is altered in CPT1C KO neurons. In parallel, we have studied the molecular mechanisms of CPT1C effect on AMPARs. We have shown that the palmitoylable cysteine 585 of the GluA1 subunit is crucial for the CPT1C enhancement of AMPA receptors trafficking by using immunofluorescence and electrophysiological techniques. However, the palmitoylation state of this residue does not determine AMPAR-CPT1C interaction. We have found evidences for a supposed depalmitoylation activity by CPT1C studying the role of the C-terminus of CPT1C, which contains the residue His469 with catalytic activity. Specifically we have found that CPT1C H469A mutant form does not alter GluA1 induced whole-cell currents as the wild type CPT1C does indicating that the C-terminal catalytic domain plays a crucial role in GluA1 modulation. This is supported by the fact that Palmostatin B – a newly described palmitoyl thioesterase inhibitor – decreases the CPT1C effect on GluA1 induced currents, most likely by inhibiting its PTE activity. In summary, this thesis unravels a novel regulation of AMPA receptor function by the interacting protein CPT1C, which modulates AMPA receptor trafficking and this effect depends on the catalytic domain of CPT1C C-terminal acting on the cysteine 585 of GluA1 AMPAR subunit.[spa] Los receptores de glutamato tipo AMPA son fundamentales en la transmisión excitatoria rápida que se da en el sistema nervioso central. A parte de su papel crucial en la comunicación neuronal, los receptores AMPA son responsables de ciertos tipos de plasticidad sináptica, siendo importantes en el desarrollo del sistema nervioso central además de estar involucrados en multitud de procesos patológicos y enfermedades neurodegenerativas. La función de los receptores AMPA depende principalmente de dos factores: la composición de las subunidades que lo conforman (el receptor propiamente dicho) y la presencia de proteínas que interactúan con el receptor y actúan como subunidades auxiliares. Estos dos factores establecen las características intrínsecas del canal así como las interacciones de los receptores AMPA con otras proteínas intracelulares que determinarán sus propiedades de tráfico (ensamblaje, exocitosis, endocitosis y anclaje sináptico) y en último término sus funciones en la neurona. Entre la gran cantidad de proteínas que interaccionan con los receptores AMPA, recientes estudios proteómicos han demostrado que la proteína CPT1C forma parte del conjunto macromolecular de los receptores AMPA en el tejido neuronal. El objetivo principal de esta tesis se ha focalizado en el estudio esta proteína (CPT1C) en el contexto de la función de los receptores AMPA debido a su interacción. Para ello se han utilizado técnicas electrofisiológicas, de inmunofluorescencia y de biología molecular y celular tanto en sistemas de expresión heterólogos como en cultivos de células neuronales. Los experimentos llevados al cabo durante la tesis han confirmado la interacción entre las dos proteínas y han atribuido un papel modulador de CPT1C en el trafico de los receptores AMPA. El efecto regulador de la CPT1C es dependiente de la composición del receptor, afectando de manera diferencial subtipos distintos de receptores AMPA. La proteína CPT1C aumenta el tráfico de los receptores AMPA a la superficie celular sin alterar sus propiedades biofísicas y sin interaccionar a nivel de membrana plasmática ambas proteínas. Además en esta tesis se han descrito los mecanismos implicados en la modulación de dichos receptores por parte de CPT1C, desentrañando un residuo cisteína concreto determinante para la modulación de los receptores AMPA por la CPT1C. Finalmente, los experimentos llevados a cabo en esta tesis nos indican que los efectos observados se deben a una posible depalmitoilación del receptor, lo cual lleva a un aumento de su capacidad de acumulación en la membrana. En resumen, los resultados obtenidos durante esta tesis demuestran que el número de receptores AMPA en las sinapsis está aumentado en presencia de CPT1C y este efecto es debido a la actividad catalítica de CPT1C

Technology of biogeometric profile control of children posture in senior preschool age during physical rehabilitation process

Actuality. One of the main determinants of the effectiveness of physical rehabilitation process of children 5-6 years old with functional disorders of the musculoskeletal system is the regular monitoring of biogeometric profile state of their posture. Only in this case, it is possible to receive feedback, timely correction and direction of the physical rehabilitation process in order to achieve the main goal - to obtain the maximum health effect. The research objective is to develop a monitoring technology for the biogeometric profile of children posture in 5-6 years old during the physical rehabilitation process, in the conditions of pre-school educational establishments. Research results. The monitoring technology for the biogeometric profile state of posture of children 5-6 years old is experimentally grounded basing on the research results. This technology consists of information-methodical, diagnostic, analytical and prognostic correction-prophylactic blocks and of two modules - express-controlling and analytical. The developed technology for monitoring the level of the biogeometric profile state of children posture of 5-6 years old contains methodological and organizational approaches and allows us to assess the adequacy of correctional and prophylactic measures and to introduce differentiated health programs into the physical rehabilitation practice, basing on the received quantitative data. Conclusions. The diagnostic technology of the biogeometric profile state of the child’s posture can be used during the physical rehabilitation process in the following areas: the development of individual and group programs for correction of disorders in the body spatial organization of children 5-6 years old; providing the necessary geometry of the masses of the whole body and its individual biolinks; development of recommendations for the rational use of motion inertia of the mass of the whole body of children 5-6 years old and the masses of its individual biolinks in order to economize various motor actions and to solve various motor tasks effectively, for example, the formation of static dynamical posture. Prospects for further research are related to the development and implementation of the concept on correction and prevention of functional disorders of child’s musculoskeletal system in the senior preschool age during physical rehabilitation process

Use of correctional technologies in the process of health-recreational fitness training by men with impaired biogeometric profile of posture

The purpose of the article is to study the effectiveness of correctional technology applied for impairment of biogeometric profile of posture observed among mature men in the process of their health-recreational fitness training. Objective. In general, the number of respondents involved in the sequential transformation phase of the experiment comprised of n = 50 people, i. e. men in the second period of their adulthood (35 – 45 years). Methods. The paper uses the data gained from the analysis of professional scientific and methodological literature, pedagogical observation; functional assessment of movements; analysis of men's posture was performed using the "Torso" program, visual screening of the biogeometric profile of posture, methods of mathematical statistics. Results of the study. The paper presents a theoretical generalization as well as it offers a new solution to the scientific task, which presupposes development and scientific substantiation of correctional technology, applied for impairment of biogeometric profile of posture observed among men of the second period of their adulthood in the process of their health-recreational fitness training, all together aiming at improving general health. The author's technology consisted of three stages, in which physical activity was strictly regulated. The letter was determined on the basis of diagnosing the state of men's posture biogeometric profile. Among the criteria for the effectiveness of the author's technology the following ones should be noted, including the type of posture, the level of biogeometric profile of posture, the level of movements functional assessment, indicators of physical development, as well as physical fitness level of men aged 36 – 45 years. Conclusions. The results of a consistent transformation experiment have confirmed the effectiveness of the developed technology when it is used for correction of impaired biogeometric profile of the posture observed among men in the second period of their adulthood during their fitness training process. During the study the effectiveness of the author's technology was experimentally confirmed, which in its turn served as a basis for recommending it for practical use in the process of health-recreational fitness training by mature men. The framework of this work does not reduce the scientific potential of the chosen issue, and therefore the prospect of further research is to introduce and study the effectiveness of a differentiated approach to health-recreational fitness training by mature women given the level of biogeometric profile of their posture

Characteristics of biomechanical properties of child's foot 5-6 years old in the physical rehabilitation process

Actuality. Pre-school age is the most responsible period in the physical development process and in a child's personality formation. The disorders in the motor function of the foot are the most common among the various pathologies of a child’s lower extremities. Many researchers have found that unfixed disorders with child's foot can eventually lead to serious changes throughout the organism and cause pathology. The study of the foot support-spring function peculiarities of children 5-6 years old in the physical rehabilitation process is not sufficiently highlighted in the available special literature. Research objective: to study the support-spring properties of a child’s foot 5-6 years old as a development precondition for the technology of the correction of unfixed disorders in children’s support-motional apparatus with physical rehabilitation using. Research results. It was established that the foot length of preschool children was within the age norm, the minimum length was 135.1 mm, and the maximum one was 201.2 mm. The obtained average statistical data of the metatarsal angle α ° and the heel angle β ° in children 5-6 years old indicate the heterogeneity of the group; the range of the metatarsal angle was in the interval from 0.24° tо 21.52° and the range of the heel angle was from 12.46° to 32.38 ° respectively. We have not established statistically significant differences at the level p> 0.05 in all indicators of the foot support-spring functions of children 5-6 years old with posture disorders and with normal posture. Conclusions. There are three main functions which characterize a normal foot: spring, balancing and pushing. The development process of the support-motional apparatus of children 5-6 years old is influenced by various factors and is subjected to some changes, including pathological. The disorders of the foot motor function occupy the first place among the various pathologies of the lower extremities in children of preschool age. It is known that, in the preschool age, the foot is in the stage of intensive development; its formation has not yet been completed, so any adverse actions can lead to the certain functional changes. Prospects for further research are related to the development and implementation of the concept for the correction and prevention of functional disorders in the support-motional apparatus of senior preschool aged children in the physical rehabilitation process

Theoretical and methodological foundations of the physical rehabilitation technology of children 5-6 years old, with functional disorders of the support-motional apparatus

Actuality. Preschool age is an important stage in the child's personality formation, in the motor skills formation, in the development of physical qualities and in the provision of conditions for normal biological development. The child's organism is subjected and varied to various external factors, both negative and positive, which have a significant influence on its development. The most important factor of external influence on the child’s organism is regular physical exercises, and the exercises of physical culture which are built methodically correctly affect favorably their physical development and health promotion. Research objective: to develop a technology for the prevention and correction of non-fixed disorders in support-motional apparatus of children aged 5–6 years with physical rehabilitation using under the conditions of preschool educational establishments. Research results. The technology was developed taking into account the individual child’s characteristics: the significance level and plane of posture disorder, the peculiarities of the varus or valgus deformity of the lower extremities and the flatfoot form, the consistent patterns between them; results of test tasks; associated diseases; the ability to learn new exercises, which allows to increase the effectiveness of the impact and to reduce the volume of applied means. The functional diagram of the prevention and correction technology of non-fixed disorders in child’s support-motional apparatus in senior preschool age makes it possible to display it from the position of a system-structural approach. The physical rehabilitation technology in the conditions of preschool educational institutions for children 5-6 years old includes theoretical lessons (thematic fairy tales), morning hygienic gymnastics, correctional exercises as part of physical education lesson, physical cultural pauses, therapeutic gymnastics, massage and physical therapy elements. All impacts were carried out in the game form. Conclusions. The physical rehabilitation technology for children 5-6 years old with non-fixed disorders in support-motional apparatus is built with consideration of the variable and basic components depending on the revealed correlation interconnection between posture disorders in the frontal and sagittal planes and spring-support function of the feet. It allows to differentiate the program of rehabilitation measures and to reduce significantly their volume without loss of efficiency. Prospects for further research are related to the development and implementation of the correction and prevention concept of functional disorders within the child’s support-motional apparatus in senior preschool age during the physical rehabilitation process

Оцінювання стану постави студентів у процесі фізичного виховання на основі візуального скринінгу

Анализ данных научной и методической литературы позволяет утверждать, что уровень здоровья студенческой молодежи Украины вызывает обоснованную тревогу. Количество студентов, имеющих отклонения в состоянии здоровья и физического развития, увеличивается с каждым годом. Такая ситуация обусловлена не только информационным и эмоциональным перенапряжением, которые влияют на студентов в процессе обучения, но и социально-экономическими, экологическими проблемами, а также дефицитом двигательной активности студентов. Как следствие этого – увеличение числа молодежи с нарушениями биогеометрического профиля осанки. Цель работы – определить особенности биогеометрического профиля осанки студентов в процессе физического воспитания на основе использования визуального скрининга. В исследовании принимали участие 250 студентов. Установлено, что только 15, 2 % студентов имеют нормальную осанку, а наибольшее количество нарушений осанки наблюдается во фронтальной плоскости: сколиотическая осанка выявлена у 36,4 % обследуемых. В сагиттальной плоскости наблюдаются следующие типы нарушений осанки: круглая спина – у 24, 4 % обследуемых, сутулая спина – у 24 % студентов. В результате исследования также выявлено, что 30 % студентов с нормальной осанкой входят в «зону риска» возникновения нефиксированных нарушений опорно-двигательного аппарата.Analysis of scientific and methodical literature states that health of studentsin Ukraine is a matter of concern. The number of students with deviations in stateof health and physical development increases every year. This situation is causednot only by information and emotional tension that affect students in thelearning process, but also by socio-economic and environmental problems and bydeficiency of motor activity of students. A consequence is an increasing numberof young people with impaired bio geometric side view of posture. The Purposeof the work is to determine the characteristics of bio geometric side view of postureof students in the process of physical education through the use of visualscreening. The study involved 250 students. It was found that only 15, 2% ofthe students have a normal posture and the greatest number of irregularities inposture is in the frontal plane: scoliosis was detected in 36.4% of surveyed. The following types of posture can be observed in the sagittal plane: 24,4% ofsurveyed have round back, 24% of students have huched back. The study alsofound that 30% of students with a normal posture are in the "dangerzone" of occurrence of deviations of Musculoskeletal system.Аналіз даних наукової та методичної літератури дає змогу стверджувати, що рівень здоров'я студентської молоді України викликає обґрунтовану тривогу. Кількість студентів що мають проблеми зі здоров'ям і низький фізичний розвиток, збільшується з кожним роком. Така ситуація зумовлена не лише інформаційним і емоційним перенапруженням, які впливають на студентів у процесі навчання, але і соціально-економічними, екологічними проблемами, а також дефіцитом рухової активності студентів. Як наслідок цього – збільшення кількості молоді з порушеннями біогеометричного профілю постави. Мета роботи – визначити особливості біогеометричного профілю постави студентів у процесі фізичного виховання на основі використання візуального скринінгу. У дослідженні брали участь 250 студентів. Встановлено, що лише 15, 2 % студентів мають нормальну поставу, а найбільшу кількість порушень постави простежено у фронтальній площині: сколіотичну поставу виявлено у 36,4 % обстежуваних. У сагітальній площині наявні такі типи порушень постави: кругла спина – в 24, 4 % обстежуваних, сутула спина – у 24 % студентів. У результаті дослідження також виявлено, що 30 % студентів з нормальною поставою належать до «зони ризику» виникнення нефіксованих порушень опорно-рухового апарату

AMPAR/TARP stoichiometry differentially modulates channel properties

AMPARs control fast synaptic communication between neurons and their function relies on auxiliary subunits, which importantly modulate channel properties. Although it has been suggested that AMPARs can bind to TARPs with variable stoichiometry, little is known about the effect that this stoichiometry exerts on certain AMPAR properties. Here we have found that AMPARs show a clear stoichiometry-dependent modulation by the prototypical TARP γ2 although the receptor still needs to be fully saturated with γ2 to show some typical TARP-induced characteristics (i.e. an increase in channel conductance). We also uncovered important differences in the stoichiometric modulation between calcium-permeable and calcium-impermeable AMPARs. Moreover, in heteromeric AMPARs, γ2 positioning in the complex is important to exert certain TARP-dependent features. Finally, by comparing data from recombinant receptors with endogenous AMPAR currents from mouse cerebellar granule cells, we have determined a likely presence of two γ2 molecules at somatic receptors in this cell type

AMPAR interacting protein CPT1C enhances surface expression of GluA1-containing receptors

AMPARs mediate the vast majority of fast excitatory synaptic transmission in the brain and their biophysical and trafficking properties depend on their subunit composition and on several posttranscriptional and posttranslational modifications. Additionally, in the brain AMPARs associate with auxiliary subunits, which modify the properties of the receptors. Despite the abundance of AMPAR partners, recent proteomic studies have revealed even more interacting proteins that could potentially be involved in AMPAR regulation. Amongst these, carnitine palmitoyltransferase 1C (CPT1C) has been demonstrated to form an integral part of native AMPAR complexes in brain tissue extracts. Thus, we aimed to investigate whether CPT1C might be able to modulate AMPAR function. Firstly, we confirmed that CPT1C is an interacting protein of AMPARs in heterologous expression systems. Secondly, CPT1C enhanced whole-cell currents of GluA1 homomeric and GluA1/GluA2 heteromeric receptors. However, CPT1C does not alter the biophysical properties of AMPARs and co-localization experiments revealed that AMPARs and CPT1C are not associated at the plasma membrane despite a strong level of co-localization at the intracellular level. We established that increased surface GluA1 receptor number was responsible for the enhanced AMPAR mediated currents in the presence of CPT1C. Additionally, we revealed that the palmitoylable residue C585 of GluA1 is important in the enhancement of AMPAR trafficking to the cell surface by CPT1C. Nevertheless, despite its potential as a depalmitoylating enzyme, CPT1C does not affect the palmitoylation state of GluA1. To sum up, this work suggests that CPT1C plays a role as a novel regulator of AMPAR surface expression in neurons. Fine modulation of AMPAR membrane trafficking is fundamental in normal synaptic activity and in plasticity processes and CPT1C is therefore a putative candidate to regulate neuronal AMPAR physiology

Novel Regulation of the Synthesis of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor Subunit GluA1 by Carnitine Palmitoyltransferase 1C (CPT1C) in the Hippocampus

The regulation of AMPA-type receptor (AMPAR) abundance in the postsynaptic membrane is an important mechanism involved in learning and memory formation. Recent data suggest that one of the constituents of the AMPAR complex is carnitine palmitoyltransferase 1C (CPT1C), a brain-specific isoform located in the endoplasmic reticulum of neurons. Previous results had demonstrated that CPT1C deficiency disrupted spine maturation in hippocampal neurons and impaired spatial learning, but the role of CPT1C in AMPAR physiology had remained mostly unknown. In the present study, we show that CPT1C binds GluA1 and GluA2 and that the three proteins have the same expression profile during neuronal maturation. Moreover, in hippocampal neurons of CPT1C KO mice, AMPAR-mediated miniature excitatory postsynaptic currents and synaptic levels of AMPAR subunits GluA1 and GluA2 are significantly reduced. We show that AMPAR expression is dependent on CPT1C levels because total protein levels of GluA1 and GluA2 are decreased in CPT1C KO neurons and are increased in CPT1C-overexpressing neurons, whereas other synaptic proteins remain unaltered. Notably, mRNA levels of AMPARs remained unchanged in those cultures, indicating that CPT1C is post-transcriptionally involved. We demonstrate that CPT1C is directly involved in the de novo synthesis of GluA1 and not in protein degradation. Moreover, in CPT1C KO cultured neurons, GluA1 synthesis after chemical long term depression was clearly diminished, and brain-derived neurotrophic factor treatment was unable to phosphorylate the mammalian target of rapamycin (mTOR) and stimulate GluA1 protein synthesis. These data newly identify CPT1C as a regulator of AMPAR translation efficiency and therefore also synaptic function in the hippocampus

Mechanisms of CPT1C-Dependent AMPAR trafficking enhancement

In neurons, AMPA receptor (AMPAR) function depends essentially on their constituent components:the ion channel forming subunits and ion channel associated proteins. On the other hand, AMPAR trafficking is tightly regulated by a vast number of intracellular neuronal proteins that bind to AMPAR subunits. It has been recently shown that the interaction between the GluA1 subunit of AMPARs and carnitine palmitoyltransferase 1C (CPT1C), a novel protein partner of AMPARs, is important in modulating surface expression of these ionotropic glutamate receptors. Indeed, synaptic transmission in CPT1C knockout (KO) mice is diminished supporting a positive trafficking role for that protein. However, the molecular mechanisms of such modulation remain unknown although a putative role of CPT1C in depalmitoylating GluA1 has been hypothesized. Here, we explore that possibility and show that CPT1C effect on AMPARs is likely due to changes in the palmitoylation state of GluA1. Based on in silico analysis, Ser 252, His 470 and Asp 474 are predicted to be the catalytic triad responsible for CPT1C palmitoyl thioesterase (PTE) activity. When these residues are mutated or when PTE activity is inhibited, the CPT1C effect on AMPAR trafficking is abolished, validating the CPT1C catalytic triad as being responsible for PTE activity on AMPAR. Moreover, the histidine residue (His 470) of CPT1C is crucial for the increase in GluA1 surface expression in neurons and the H470A mutation impairs the depalmitoylating catalytic activity of CPT1C. Finally, we show that CPT1C effect seems to be specific for this CPT1 isoform and it takes place solely at endoplasmic reticulum (ER). This work adds another facet to the impressive degree of molecular mechanisms regulating AMPAR physiology