Thèses sur le sujet « Neurones NPY »

Dans le hilus du gyrus denté de l'hippocampe, les cellules moussues excitatrices et les interneurones GABAergiques constituent des acteurs clés du réseau. Cependant, en raison en partie de leur grande diversité, la fonction des interneurones GABAergiques du hilus dans la physiologie du gyrus denté reste peu détaillée. Nous avons utilisé des souris transgéniques GAD67-GFP, exprimant la GFP sous le contrôle du promoteur de la GAD67, et évalué les densités de neurones GABAergiques marqués pour la Calretinine (CR), Parvalbumine (PV), Somatostatine (SOM), Neuropeptide Y (NPY) et l'oxyde nitrique synthase (NOS-1) dans le hilus et la couche granulaire. Pour mieux caractériser les différentes populations d'interneurones, nous avons caractérisé les propriétés de 123 neurones en utilisant la technique de RT-PCR sur cellule unique sur des tranches de cerveaux de souris C57Bl6 âgées de 2 à 3 mois. Une analyse non supervisée en clusters basée sur 18 paramètres électrophysiologiques et 7 paramètres moléculaires a clairement mis en évidence un cluster de cellules moussues excitatrices (n=67) et 3 clusters de cellules GABAergiques (n=56). Les deux premiers clusters d'interneurones GABAergiques comprennent des neurones (n=18 et n=16) qui co-expriment le NPY et la SOM mais se différencient clairement par leurs propriétés élcetrophysiologiques. Le troisième cluster d'interneurones comprend des neurones à décharge rapide exprimant soit la PV (n=9) soit la NOS-1 (n=13). Cette caractérisation multiparamétrique supporte l'existence de classes fonctionnelles distinctes parmi les interneurones GABAergiques du gyrus denté

Le glucose, en plus d'être un substrat métabolique, agit en tant que molécule informative en modulant l'activité de certains neurones dits " sensibles au glucose ". Nos enregistrements électrophysiologiques menés dans le noyau arqué de souris montrent que 4 sous-populations de neurones sont présentes dans ce noyau. Les neurones dits GE et GI sont respectivement excités et inhibés par une hypoglycémie (5-0 mM de glucose) ; les neurones HGE et HGI sont respectivement excités et inhibés par une hyperglycémie (5-20 mM de glucose). Nous avons entrepris de déterminer la nature des ces neurones sensibles au glucose en étudiant les neurotransmetteurs qu'ils expriment. En utilisant des souris NPY-GFP, nous montrons que les neurones GI sont des neurones à NPY. L'utilisation de souris POMC-GFP montre que les neurones GE et HGE n'expriment pas la POMC. Ce résultat a été confirmé in vivo en utilisant le marqueur d'activité neuronale c-fos
Glucose is known to regulate energetic homeostasis in controlling the electrical activity of glucose-sensing neurones. Using electrophysiological recordings on mouse brain slice, we show that 4 distinct glucose-sensitive neurones subpopulations exist in the arcuate nucleus. GE and GI neurones are respectively excited and inhibited by a hypoglycaemia (5-0 mM glucose); HGE and HGI neurones are respectively excited and inhibited by a hyperglycaemia (5-20 mM glucose). Then, in order to suggest a physiologic relevance of these neurones, we decided to characterize the neuropeptides express in these cells. Using NPY-GFP mouse, we show that GI neurones express NPY. Nevertheless, using POMC-GFP mouse, we show that GE or HGE neurones do not express POMC. This result was confirmed in vivo, in using the neuronal activating marker c-fos. This work replaces these glucose-sensitive neurones in the neural network involved in the control of energetic homeostasis

La prise alimentaire est soumise à des processus de régulation complexes dans lesquels interviennent de nombreux neuropeptides hypothalamiques. Parmi eux, le neuropeptide Y (NPY) exerce un puissant effet orexigène alors que les mélanocortines, issues du clivage protéolytique de la proopiomélanocortine (POMC), et le pituitary adenylate cyclase-activating polypeptide (PACAP) diminuent la prise de nourriture. L'objectif de notre étude a été de déterminer les relations neuroanatomiques et fonctionnelles entre les neurones synthétisant la POMC dans le noyau arqué (NA) hypothalamique et les systèmes neuropeptidergiques à NPY et à PACAP. Ce travail de thèse apporte de nouvelles informations sur la contribution des neurones à POMC du NA dans la régulation de l'homéostasie énergétique. Il apparaît que les neurones à POMC sont capables de contrôler directement l'activité des neurones à NPY du NA et qu'ils sont eux-mêmes soumis à une modulation par le PACAP
The food intake is under control of a complex regulation mechanism involving several hypothalamic neuropeptides. Among them, neuropeptide Y (NPY) is orexigenic whereas α-melanocyte-stimulating hormone (α-MSH), a peptide generated by processing of proopiomelanocortin (POMC), and pituitary adenylate cyclase-activating polypeptide (PACAP) reduce food consumption. We have investigated the neuroanatomic and functional relations between POMC neurons of arcuate nucleus and the NPY and PACAP neuropeptidergic systems. Our data provide additionnal evidence for the function of arcuate POMC neurons in the regulation of energy homeostasis. Thus, it appears that POMC neurons are able to directly control the activity of the arcuate NPY system and that they are subjected to modulation by PACAP

La perturbation des mécanismes de réponse au stress chez les organismes peut entraîner une dysfonction cellulaire et des maladies telles que le syndrome métabolique. L'équilibre énergétique est principalement régulé par le système nerveux central (SNC), qui intègre des signaux hormonaux, neuronaux et alimentaires provenant de divers tissus. Une dysfonction de ce système est liée à l'obésité et au syndrome métabolique, qui sont tous deux des précurseurs du diabète de type 2 (T2D). Notre laboratoire a découvert que la protéine kinase ID dépendante du calcium/calmoduline (CaMK1D), un gène associé au T2D, favorise la prise alimentaire médiée par la ghréline chez les souris. Cependant, le rôle de la signalisation de CaMK1D dans les neurones NPY/AgRP reste encore à éclaircir. Dans cette étude, nous avons réalisé un séquençage de l'ARN en utilisant la lignée cellulaire hypothalamique GT1-7. Nous avons ainsi découvert que CalHM6 est une cible potentielle en aval de la signalisation de CaMK1D. Les niveaux d'ARNm de CalHM6 sont ainsi significativement augmentés dans les cellules CaMK1D-/- et sont réduits lorsque CaMK1D est ré-exprimé. Cela a également été confirmé in vivo dans l'hypothalamus des souris CaMK1D-/-. CalHM6, probablement un canal calcique dépendant du voltage, a montré des niveaux intracellulaires de Ca2+ augmentés en réponse à la ghréline dans les cellules CaMK1D-/- par rapport aux cellules CaMK1D+/+ en utilisant la méthode jGCamps. En résumé, notre travail a montré que CalHM6 est une nouvelle cible de CaMK1D. Une expression élevée de CaMK1D, entraînant une faible expression de CalHM6, pourrait ainsi favoriser la prise alimentaire et l'obésité en modulant la signalisation dépendante du calcium dans les neurones NPY/AgRP
Disruption of stress response mechanisms in organisms can lead to cellular dysfunction and diseases like metabolic syndrome. Energy balance is mainly regulated by the central nervous system (CNS), which integrates hormonal, neuronal, and dietary signals from various tissues. Dysfunction in this system is linked to obesity and metabolic syndrome, both precursors to type 2 diabetes (T2D). Our laboratory discovered that calcium/calmodulin-dependent protein kinase ID (CaMK1D), a gene associated with T2D, promotes ghrelin-mediated food intake in mice. However, CaMK1D signaling in NPY/AgRP neurons still remains questions. In this work, we proformed RNA sequencing using the GT1-7 hypothalamic cell line. To this end, we found that CalHM6 is a downstream target of CaMK1D signaling. CalHM6 mRNA levels were significantly upregulated in CaMK1D-/- cells and downregulated when CaMK1D was re-expressed. This was confirmed in vivo in the hypothalamus of CaMK1D-/- mice. CalHM6, likely a voltage-gated calcium channel, showed increased intracellular Ca2+ levels in response to ghrelin in CaMK1D-/- cells compared to CaMK1D+/+ cells using jGCamps method. Altogether, our work showed CalHM6 is a novel target of CaMK1D. High CaMK1D, leading to low CalHM6 expression, may enhance food intake and obesity by modulating calcium-dependent signaling in NPY/AgRP neuron

Le système nerveux central (SNC) intègre en permanence des signaux périphériques tels que les variations de concentration en hormones et en nutriments, et module en réponse le comportement alimentaire ainsi que la dépense énergétique et le métabolisme. Au niveau du noyau arqué de l'hypothalamus, la barrière hématoencéphalique est plus perméable, ce qui lui confère un rôle de premier ordre dans l'intégration des signaux périphériques. Les neurones du noyau arqué de l'hypothalamus qui sécrètent le neuropeptide Y et l'Agouti Related Protein (NPY/AgRP) sont impliqués dans la stimulation de la prise alimentaire et la diminution de la dépense énergétique. Contrairement aux résultats attendus, l'ablation de ces neurones oréxigènes au stade néonatal chez la souris conduit au développement d'une obésité massive à l'âge adulte. Cette obésité se développe sans hyperphagie, et les analyses métaboliques montrent que les animaux ont une dépense énergétique similaire aux témoins. Les souris déficientes ne sont pas diabétiques contrairement à d'autres modèles d'obésité, mais sont caractérisées par une hyperinsulinémie basale associé à une altération du métabolisme lipidique. Nous montrons que la perte de ces neurones induit une modulation du renouvellement des catécholamines qui se traduit en périphérie par une redistribution des flux métaboliques, favorisant le métabolisme lipidique. Cette redistribution serait responsable du développement de l'obésité en favorisant les mécanismes de stockage, mais permettrait de limiter les effets d'un régime gras sur l'homéostasie glucidique
Several neuronal populations are involved in the regulation of energy balance. Among these, the hypothalamic agouti-related protein neurons (AgRP-neurons) are well characterized for their ability to promote food intake. Using cell-specific ablation we investigated a possible role of AgRP neurons in nutrient partitioning independent from food intake. We show that mice lacking AgRP neuron developed a non-hyperphagic obesity due to increased feed efficiency (weight gain/kcal consumed) on regular chow. At a time that preceded obesity catecholamine turnover rate was selectively decreased in the pancreas, liver, and glycolytic muscle, while it was increased in oxidative muscle. Respiratory quotient measurements revealed a change in substrate utilization towards lipid oxidation enhanced by synergistic increase in liver triglyceride synthesis and lipid substrate preference in oxidative muscle mitochondria. On high fat diet, mice lacking AgRP neurons displayed reduced body weight gain and paradoxical improvement in glucose tolerance. Finally we evidenced that Gamma Aminobutyric Acid (GABA) made by AgRP neurons is important in the central control of peripheral substrate utilization. This study reveals a new function for AgRP neurons in the coordination, via the sympathetic nervous System, of inter-organs communication and nutrient partitioning. These results offer a new conceptual framework for the understanding of obesity-related disorders

Local inhibitory microcircuit of amygdala is an active component in processing emotional information. Despite prominent evidence of its importance, our understanding of GABAergic cell types, their connectivity and role in amygdala network is limited. The aim of this thesis is to understand connectivity and physiology of two specific components of GABAergic microcircuit of amygdala: so-called intercalated cells and neuropeptide Y (NPY) expressing interneurons. Intercalated cells (ITCs) of the amygdala are clusters of GABAergic neurons that surround the basolateral complex of amygdala (BLA). There is growing evidence suggesting that ITCs are required for the expression of fear extinction. The main intercalated nucleus (Im) is the largest of the ITC clusters and could be also important for emotional processing. Using patch-clamp whole-cell recordings paired with subsequent anatomical analysis I described basic physiology and anatomy of neurons within the Im. I found that these neurons share common characteristics to earlier described neurons within the medial ITC cluster, yet they can be divided into three distinct groups. Next, I provided anatomical and functional evidence that Im neurons project to central and basal nucleus of amygdala and that they are reciprocally connected with medial and lateral ITCs clusters. I found that Im neurons receive excitatory inputs from BLA as well as cortex; next I verified that heterogeneous inputs do not interact with each other. I have shown that the Im neurons express both AMPA and NMDA receptors, suggesting that they may undergo NMDA-dependent plasticity. I have reported that dopamine hyperpolarizes Im neurons via dopamine receptor 1, therefore providing a cellular substrate for disinhibition of the amygdala at the systemic level. Thus, the Im is likely to be an additional site of integration of the distributed network underlying acquisition, expression and extinction of conditioned fear. In another project, I report novel interneuron type of the BLA and call it neurogliaform cell (NGFC) of amygdala. I used a mouse line expressing green fluorescent protein (GFP) under NPY promoter and patch clamp technique combined with pharmacology and electron microscope analysis. I performed paired recordings between presynaptic NPY-GFP positive (+) cells and postsynaptic principal neurons (PNs). Presynaptic NPY-GFP+ neurons display small soma and short dendrites embedded in a cloud of highly arborized axon. I showed that NPY-GFP+ cells are source of GABAA receptor-mediated slow inhibitory postsynaptic currents (IPSCs, decay time constant > 30 ms) evoked in PNs and in themselves (autapses). These slow IPSCs are known in literature as GABAA,slow. My results indicate that the slow kinetics of these IPSCs was likely caused by the low concentration and spillover of extracellular GABA. Physiologically-relevant in vivo firing re-played in NPY+-NGFCs in vitro evoked a transient depression of the IPSCs. Presynaptic GABAB receptors controlled the strength of this short-term plasticity. Interestingly, synaptic contacts made by NGFCs showed close appositions, without identifiable classical synaptic structures, between presynaptic boutons of the recorded cells and postsynaptic profiles. Thus, volume transmission of GABA is likely to be generated by this interneuron of the amygdala. NPY+-NGFC is a novel interneuron type of the BLA. The peculiar functional mode of NGFCs makes them unique amongst all GABAergic cell types of the amygdala identified so far.

Neuropeptide Y (NPY) and NPY receptors are concentrated in the hippocampal formation where they modulate cognitive functions and seizures. The cellular mechanisms underlying the physiological roles of NPY in the hippocampus are still rather elusive. Cultured hippocampal cells offer a simple model to study the expression of hippocampal NPY receptor subtypes and their respective cellular distribution.
The present thesis examines the presence of NPY receptors in primary dissociated hippocampal cells in culture using a combination of molecular, pharmacological and immunocytochemical approaches. Receptor binding experiments revealed the preferential expression of Y1-like receptors over the Y2, Y4 and Y5 subtypes in rat hippocampal cultures. Cultured hippocampal cells expressed high level of Y1 receptors and very low amounts (below detection) of the Y2, Y 4 and Y5 receptor binding sites. The genuine nature of the Y1-like receptor expressed in these cells was confirmed using amplification (RT-PCR) of Y1 receptor mRNAs. The cellular phenotype expressing Y1 receptor was investigated using double labeling methods. Specific autoradiographic and immunolabeling signals of Y1-like receptors were observed on neurons (70% of the total population of neurons) and astrocytes (20% of the total population of glia cells) as revealed respectively by neuron specific enolase (NSE) and glial fibrillary acidic protein (GFAP) immunostaining. The Y1 receptor labeling is distributed uniformly over neuronal cell bodies and processes compared to a non-uniform and clustered distribution on type I astrocytes. Neurons labeled by the various Y1 receptor markers were mostly glutamate-positive as revealed by double labeling. Interestingly, a small proportion (2--5%) of NPY-positive hippocampal neurons (NPY-positive cells represent around 10% of the total population of neurons) were also enriched with Y1 receptor labeling. These results suggest the possible autoregulatory role for the Y1 receptor on NPY release.
Taken together, these anatomical and pharmacological results suggest that Y1 receptor may play an important role in the hippocampus via the regulation of astrocytic and/or neuronal functions and the modulation of the release of glutamate and/or NPY.

Cette thèse concerne les mécanismes neuronaux impliqués dans l’intégration mathématique d’un signal de vitesse en signal de position. Dans le cadre du contrôle des mouvements horizontaux de l’œil, cette intégration est réalisée par les neurones du noyau prepositus hypoglossi (nNPH). Les nNPH ont été classés, selon leur profil électrophysiologique, en 3 types (A, B et D) et modélisés. Contrairement aux neurones de type A et B présents aussi dans les noyaux vestibulaires médian et latéral, les neurones de type D sont spécifiques du NPH et leur potentiel de membrane présente des oscillations. De plus, la conductance sodique persistante est cruciale pour l’électrophysiologie de tous les nNPH, quoique son impact et sa localisation diffèrent selon les types cellulaires. Enfin, les propriétés intrinsèques des neurones du NPH et des noyaux vestibulaires ont été comparées afin de comprendre le lien entre les fonctions de ces noyaux et les propriétés intrinsèques spécifiques de leurs neurones
The rationale behind this thesis is the understanding of the neural mechanisms involved in the mathematical integration of a velocity signal into a position signal. For eye movements in the horizontal plane, neurons of the prepositus hypoglossi nucleus (PHNn) are responsible for this integration. Here, PHNn have been classified in 3 types (A, B and D) according to their electrophysiological profile and then modeled. Unlike type A and B neurons, which are also found in the medial and lateral vestibular nuclei, type D neurons are specific to the NPH and their membrane potential shows subthreshold oscillations. Besides, persistent sodium conductance is crucial to the electrophysiology of the PHNn, however its impact and location are type-dependant. The intrinsic properties of neurons of the PHN and the vestibular nuclei have been compared to understand the link between the functions of these nuclei and the specific intrinsic properties of their respective neurons

Thèse de doctorat : Sciences de l'ingénieur. Génie informatique, automatique et traitement du signal : Paris 12 : 2007.
Thèse uniquement consultable au sein de l'Université Paris 12 (Intranet). Titre provenant de l'écran-titre. Bibliogr. : 105 réf.

Neurotoxicity is defined as “an adverse change in the structure or function of the nervous system that results from exposure to a chemical, biological or physical agent". lonic channels, transporters, receptors for neurotransmitters and neurohormones are the main modulators of neuronal activity. It is therefore to be expected that the interfering with these proteins induces significant and potentially damaging effects in these tightly regulated and highly responsive cells. The present research project has been focused on the study of the role of specific ionic channels and the main mechanisms involved regarding two cases of neurotoxicity, which were widely documented but not completely understood: 1) the potential neurotoxicity induced by SiO2 NP and 2) the one induced by OHP. This issue has been addressed by means of an integrated approach that combine electrophysiological recordings both at single cell level by using the patch clamp technique, and at population level by using multielectrode arrays (MEAs), as well as quantitative real-time polymerase chain reaction (qRT-PCR) and/or RNAseq for a trascriptome screening. Members of TRP channel family (TRPA1, TRPV1, TRPV4), two pore domain K+ (K2P) channel family, as well as connexins and pannexin-like channels, are the major components of the responses by SiO2 NP and OHP. In conclusion, the information presented here may be valuable, particularly for contributing to current knowledge on this subject mainly regarding cells of the nervous system, as some evidence and mechanistic suggestions are provided.

Tese de doutoramento em Ciências Farmacêuticas, na especialidade Farmacologia e Farmacoterapia, apresentada à Faculdade de Farmácia da Universidade de Coimbra
O hipotálamo é uma região do cérebro que regula o desenvolvimento, o crescimento e o metabolismo. Recentemente, foi também demonstrado que o hipotálamo desempenha um papel chave no desenvolvimento generalizado do envelhecimento. A autofagia é um processo intracelular envolvido na reciclagem dos constituintes da célula e na manutenção da homeostase celular. Durante o envelhecimento e em doenças associadas ao envelhecimento ocorre diminuição da autofagia. Por outro lado, em diversas espécies de animais, a restrição calórica (RC) é uma robusta intervenção anti-envelhecimento, aumentando o tempo de vida e diminuindo a incidência de doenças associadas à idade. A RC estimula a autofagia e também aumenta os níveis do neuropeptídeo Y (NPY) no hipotálamo. Diversos trabalhos mostram que o NPY tem um papel neuroprotector, aumenta a resistência ao stress. Contudo, o papel do NPY na autofagia nunca foi investigado. Desta forma, o primeiro objectivo deste trabalho foi estudar o papel do NPY na autofagia em neurónios do hipotálamo. Os resultados mostraram que o NPY estimula a autofagia numa linha de neurónios hipotalâmicos de murganho (mHypo-N42) e também em culturas primárias de células hipotalâmicas neurais diferenciadas de rato. O NPY aumentou o fluxo autofágico em neurónios do hipotálamo através da activação dos receptores Y1 ou Y5, e que activam as vias de sinalização intracelular PI3K, ERK e PKA. O efeito do NPY na autofagia num modelo in vivo também foi avaliado, através da sobre-expressão do NPY no núcleo arqueado do hipotálamo de murganhos C57BL/6, pela tecnologia de transferência génica usando vírus adenoassociados. Os resultados mostraram que o NPY também estimula a autofagia no hipotálamo in vivo. O núcleo arqueado do hipotálamo, responsável pela homeostase energética, é composto por duas populações neuronais distintas – neurónios que expressam POMC e CART, e neurónios que expressam NPY e AgRP. Estas duas populações regulam o anabolismo e catabolismo, recebendo e integrando sinais nutricionais e hormonais da periferia. Estudos recentes sugerem que a plasticidade sináptica dos circuitos hipotalâmicos envolvidos na ingestão alimentar também tem um papel na regulação da homeostase energética. Contudo, o papel da autofagia na plasticidade dos circuitos hipotalâmicos nunca foi investigado. Desta forma, o segundo objectivo deste trabalho foi investigar o papel da inibição específica de uma proteína fundamental do processo de autofagia, a Atg7, na organização sináptica, com uma dieta normal e em privação de alimentos. Os murganhos com inibição específica da proteína Atg7 nos neurónios POMC (POMC-Cre; Atg7loxP/loxP) foram usados como modelo animal de estudo e os murganhos com expressão inalterada de Atg7 (Atg7loxP/loxP mice) como controlos. Nestes animais avaliou-se a organização sináptica dos neurónios POMC. Os animais foram mantidos durante cerca de 10 semanas com acesso livre a uma dieta padrão ou sem acesso a comida durante uma noite. O núcleo arqueado do hipotálamo destes animais foi analisado por microscopia electrónica, microscopia de fluorescência e por microscopia óptica de luz visível. Os neurónios hipotalâmicos POMC dos murganhos POMC-Cre; Atg7loxP/loxP, com ausência de Atg7 nos neurónios hipotalâmicos POMC, apresentaram um aumento da área e do perímetro desses neurónios, e apresentaram acumulação de nematossomas. Além disso, os neurónios hipotalâmicos POMC dos murganhos POMC-Cre; Atg7loxP/loxP apresentaram mais contactos sinápticos, que se traduzem num aumento dos contactos simétricos inibitórios. Depois de uma noite sem acesso a comida, os neurónios do núcleo arqueado do hipotálamo dos murganhos POMC-Cre; Atg7loxP/loxP apresentaram menor imunorreactividade para c-Fos, que sugere menor activação neuronal. Em conclusão, os resultados desta tese mostram que o NPY induz o fluxo autofágico em neurónios do hipotálamo, e que a autofagia desempenha um papel na regulação da plasticidade sináptica dos neurónios POMC. Uma vez que a autofagia no hipotálamo e os níveis do NPY diminuem com o envelhecimento, a modulação do NPY pode ser um mecanismo protector contra a disfunção hipotalâmica associada ao aumento da idade. Por outro lado, a modulação da autofagia, através de um mecanismo sináptico subjacente, pode oferecer estratégias para a regulação do peso corporal.
The hypothalamus is the brain region that regulates development, growth and metabolism, and has gained increased attention for its key role in the progression of whole body aging. Additionally, autophagy, a highly regulated intracellular process involved in the turnover of most cellular constituents and in the maintenance of cellular homeostasis, is impaired in aging, contributing to the aging phenotype and to the aggravation of age-related diseases. On the other hand, caloric restriction (CR) is a robust anti-aging intervention, increasing lifespan and decreasing the incidence of age-related diseases. CR increases autophagy in different brain areas and increases neuropeptide Y (NPY) levels in the hypothalamus. Moroever, NPY has neuroprotective effects and increases resistance to stress and mean lifespan. However, the role of NPY on autophagy has never investigated before. Therefore, the first aim of this study was to investigate the role of NPY on autophagy in hypothalamic neurons. The results show that NPY stimulated autophagy in mouse hypothalamic cell line N42 (mHypo-N42) and also in rat differentiated hypothalamic neural cell cultures. Moreover, NPY stimulated the autophagic flux in hypothalamic neurons by activating NPY Y1 or Y5 receptors, through PI3K, ERK and PKA intracellular signaling pathways. We also evaluated the role of NPY on autophagy in vivo, by overexpressing NPY in the arcute nucleus (ARC) of hypothalamus of C57BL/6 mice, using adenoassociated viral (AAV) gene transfer technology. The results show that NPY also stimulated autophagy in hypothalamus in vivo. The hypothalamic ARC, responsible for energy homeostasis, is composed by two major neuronal populations – cocaine- and amphetamine-regulated transcript (CART)/Pro-opiomelanocortin (POMC) expressing neurons and agouti-related peptide (AgRP)/ neuropeptide Y (NPY) expressing neurons. These two neuronal populations regulate anabolic and catabolic state, receiving and integrating peripheral nutritional and hormonal signals. Recent observations suggest that synaptic plasticity in the hypothalamic feeding circuits has also a critical role in regulation of energy homeostasis, since the neuronal synaptic input organization in the hypothalamus is able to adapt and rearrange rapidly in response to metabolic hormones. In addition, autophagy in the hypothalamus was identified as a player in metabolic regulation. However, a role for autophagy in plasticity of hypothalamic feeding circuits has not been explored. Therefore, the second aim of this study was to investigate the role of Atg7 deletion in POMC neurons in the synaptic organization in mice under standard diet and food deprivation. POMC-specific Atg7 knockout mice (POMC-Cre; Atg7loxP/loxP) were used as animal model and Cre-negative Atg7loxP/loxP mice as controls, to evaluate the synaptic organization of the hypothalamic POMC neurons and neuronal activation in hypothalamic ARC. Animals were maintained during 10 weeks with standard diet or overnight fasting, and then brains slices containing arcuate nucleus of the hypothalamus were stained for electron microscopy and for fluorescence and light microscopy. The specific Atg7 deletion in POMC neurons resulted in an increased cell area and perimeter, and nematosomes accumulation. Moreover, we observed that POMC-Cre; Atg7loxP/loxP neurons have more synaptic inputs and more symmetric, putatively inhibitory inputs. After an overnight fasting, POMC-specific Atg7 knockout mice show no normal adaptation to food deprivation, with an impaired neuronal activation in hypothalamic ARC. Overall, these results show that NPY induces autophagic flux in hypothalamic neurons, and that autophagy has a role in the control of synaptic plasticity of POMC neurons. Since both hypothalamic autophagy and NPY levels decrease with age, modulation of NPY may act as a protective mechanism against impaired hypothalamic dysfunction associated with age. Moreover, autophagy modulation, through underlying synaptic mechanism, might offer strategies to the body weight regulation.
FCT - SFRH/BD/73004/2010

The abuse of new psychoactive substances (NPS) represents a threat to public health. Emergent NPS N-ethylhexedrone (NEH), N-ethylpentylone (NEP) and 4-chloroethcathinone (4-CEC) are among the most harmful and seized synthetic cathinones, a prominent NPS class. We previously showed that NEH decreases neuronal and microglial viabilities. However, studies on neuroinflammatory processes and intra-/inter-cellular signalling dysregulation by synthetic cathinones are scarce. In this thesis, we aimed to evaluate how: (i) these substances trigger inflammation and disrupt cellular homeostasis in vitro; and (ii) cathinone-exposed microglia impact neuronal function. For that, we used differentiated SH-SY5Y (neuronal) and CHME3 (microglial) human cell lines exposed to (i) either 100 or 400 μM of each NPS or (ii) secretome from exposed microglia for 24 h, using non-exposed cells as control. We demonstrated that cathinone exposure causes neuronal and microglial dysfunction, namely: (i) increased cell demise, via apoptosis and necrosis; (ii) mitochondrial impairment; and (iii) lysosomal distress. Neuroimmune responses, marked by the upregulation of several pro-/anti-inflammatory markers (S100B, iNOS, nNOS, TNF-α, IL-1β, IL-10) were observed on cells exposed to NPS. Additionally, cathinones dysregulated the genes encoding for synaptic proteins synaptophysin and Dlg4 in neurons; in microglia, these substances caused the overexpression of phagocytosis-related markers TREM2 and MFG-E8 and increased phagocytic activity (non-specific phagocytosis). We also demonstrated that the secretome from NPS-exposed microglia disturbed the neuronal immunoregulatory response and dysregulated synaptic protein plasticity, suggesting that cathinone-activated microglia may negatively impact neuronal function via paracrine signalling. Taken together, our findings highlight the ability of prevalent synthetic cathinones to trigger neuronal and microglial disabilities and homeostatic imbalance, while altering synaptic plasticity and causing inflammation that may precede (and contribute to) the onset of neurotoxic events, ultimately compromising brain function. Our data shines new light on the general and specific mechanisms underlying the detrimental impact of synthetic cathinones in human brain health.

碩士
國立陽明大學
微生物及免疫學研究所
99
Cerebrovascular disease resulting in stroke is a common cause of morbidity and mortality. In Taiwan, stroke is the third cause of death. Until now, we still don’t have a therapy that can cure stroke efficiently. The responses in brain after stroke are involves in lots of pathophysiological mechanism such as excitotoxicity, free radical damage, and inflammation. It is difficult to recover such injury via single therapy. The stem cell therapies are based on the differential potential of stem cell to achieve the purpose of tissue replacement. Beside, lots kind of stem cells have been reported having the neuroprotective capacity. Recent study implies that angiogenesis have an important role in brain tissue replacement and the endothelial progenitor cell (EPC) predominant angiogenesis in adult especially in pathological condition. In this study, we first performed the migration assay of endothelial cell to evaluate the angiogenic capacity of variant stem cells. Then we demonstrated that induced-pluripotency stem cell derived neural progenitor cell (iPSN) can secret FGF8 to attract EPC and promote angiogenesis and iPSN induce angiogenesis in MCAO rat as well. Further, downstream of FGF8 the ERK signal is essential for EPC angiogenesis. Additionally, we prove that EPC have more neuroprotective capacity then mature endothelial cell in vitro.

En este trabajo, se presenta un algoritmo para estimación de tasa de precipitación a partir de datos provenientes del sensor ATMS a bordo de la plataforma espacial Suomi-NPP. El algoritmo aprovecha la capacidad de penetración en las nubes asociada a las microodas pasivas, así como también la sensibilidad ante precipitaciones, cristales de hielo y nubes de agua precipitable, mediante el entrenamiento de redes neuronales. Con este fin se entrenan 4 redes neuronales, 2 para tierra y 2 para océano, de las cuales 1 corresponde a periodo de verano y la otra a invierno respectivamente, haciendo uso de datos simulados para los 22 canales que conforman el sensor ATMS. El algoritmo demuestra alto potencial para reproducir patrones de precipitación, así como una capacidad satisfactoria para la estimación de la magnitud de tasa de precipitación.

Niemann-Pick Type C (NPC) disease is an autosomal recessive disorder that results in accumulation of unesterified cholesterol in late endosomes/lysosomes (LE/Ls), leading to progressive neurodegeneration and premature death. Microglia are resident immune cells of the central nervous system, which upon activation can secrete potentially neurotoxic molecules such as tumor necrosis factor-alpha (TNFα). Inappropriate activation of microglia has been implicated in NPC disease. Primary microglia cultures from the cerebral cortex of Npc1-/- mice have an altered cholesterol distribution characteristic of NPC-deficient cells. Immunocytochemical analysis revealed increased TNFα staining in Npc1-/- microglia. However, Npc1-/- and Npc1+/+ microglia showed similar mRNA levels of pro-inflammatory cytokines and similar levels of TNFα secretion. To determine whether Npc1-/- microglia contribute to neuron death in NPC disease, microglia were co-cultured with cerebellar granule cells. Surprisingly, the extent of neuronal death was the same in neurons cultured with Npc1+/+ or Npc1-/- microglia. Thus, Npc1-/- microglia have an altered phenotype compared to Npc1+/+ microglia, but this does not lead to neuron death in an in vitro co-culture system. Treatment options for NPC disease remain limited. A consequence of cholesterol sequestration in the LE/Ls, is that cholesterol movement to the endoplasmic reticulum, where cholesterol metabolism is regulated, is impaired. Cyclodextrin (CD), a compound that binds cholesterol, has recently been found to delay the onset of neurological symptoms and prolong life of Npc1-/- mice. Since the brain consists of both neurons and glia, it remains unclear if CD acts directly on neurons and/or other cells in the brain. Neurons cultured from the cerebellum and astrocytes cultured from the cortex of Npc1-/- mice were treated with a low dose (0.1mM) of CD. This treatment decreased cholesterol sequestration and decreased the rate of cholesterol synthesis in Npc1-/- neurons and astrocytes. CD also decreased mRNAs encoding proteins involved in cholesterol synthesis in Npc1-/- neurons and increased genes involved in cholesterol efflux in Npc1-/- astrocytes. Moreover, CD increased cholesterol esterification in Npc1-/- astrocytes. These results suggest that cholesterol trapped in LE/Ls in Npc1-/- neurons and astrocytes was released by CD treatment and reached the ER, thereby regulating cholesterol homeostasis.
Experimental Medicine

碩士
國立中正大學
化學工程所
96
The purse of the study was to investigate the electrophoretic mobility and zeta potential of rat bone marrow stromal cells (rBMSCs) with different concertration of neuron growth factor (NGF) at various induced time, heparin loading poly (D,L lactide-co-glycolide) nanoparticles (H-PLGA NPs) prepared by different ratio of surfactant (lecithin/Tween 80) with various concertration of heparin and rBMSCs coculture with various heparin loading efficiency H-PLGA NPs at various induced time. The size of H-PLGA NPs were measured by zetasizer and confirmed by the images of FE-SEM and TEM. The fixed charge density of rBMSCs were estimated from the electrophoretic mobility of capillary electrophoresis with the soft particle electrokinetic theory. The result revealed that, the ratio of lecithin and the concertration of heparin increased, the size and the absolute value of electrophoretic mobility and zeta pontential of H-PLGA NPs increased. The absolute value of electrophoretic mobility and zeta pontential of rBMSCs increased with an increase of induced time in the process differentiation into neuron. The weight of H-PLGA NPs uptaked by rBMSCs inereased with an increase of heparin loading efficiency of H-PLGA NPs. The absolute value of electrophoretic mobility and zeta pontential of rBMSCs decreased with an increase of rBMSCs uptakes H-PLGA NPs in the process differentiation into neuron.