Opioids are gold-standard analgesics for pain relief in chronic pain conditions. Paradoxically, chronic opioid use causes an enhanced pain sensitivity termed ‘Opioid-induced hyperalgesia’ (OIH). OIH is a clinically relevant problem associated with the use of opioids. In addition to decreasing quality of life, increased pain from OIH necessitates increasing dosages of analgesics to effectively control the pain, resulting in an increased risk of opioid epidemics, addiction, and overdose. To prevent this clinically important effect, it is necessary to understand how chronic opioid use causes hyperalgesia. Our preliminary studies revealed that synaptic plasticity in the spinal dorsal horn (SDH) is dependent on neuron type in the OIH model and occurs concurrently with hyperalgesia, suggesting central sensitization as a mechanism of OIH. We found that astrocyte ablation blocked mechanical hyperalgesia and neuron type-dependent synaptic plasticity, indicating that astrocytes are critically involved in OIH. Additionally, morphine treatment upregulated IL-1β expression in the SDH in our preliminary experiments. Inhibition of IL-1β prevented OIH and blocked the repeated morphine-induced synaptic plasticity in the SDH, suggesting IL-1β is a key player in the pathogenesis of OIH. Astrocytes and other glial cells are critical in the development and maintenance of neuroinflammatory conditions, such as OIH, through the release of proinflammatory cytokines (PICs), including IL-1β. The mechanosensitive ion channel, Piezo1, was recently found to be upregulated in astrocytes and microglia under LPS-induced inflammatory conditions, and activation of Piezo1 was found to reduce IL-1β expression in LPS-inflamed primary mouse astrocytes. The goal of this study was to investigate the function of Piezo1 as a potential treatment for neuroinflammatory diseases of the CNS in a model of LPS-induced inflammation. In this study, we created a culture cell model of LPS-induced astrocytic neuroinflammation using the C8-S type II astrocyte culture cell line. We used a multi-disciplinary approach of electrophysiology and imaging to assess changes in calcium flux induced by the selective Piezo1 agonist, Yoda1, and mechanosensitive ion channel activity in the LPS-stimulated C8-S culture astrocytes. We found that calcium flux is increased in LPS stimulation and augmented by additional Yoda1 treatment. We also found that LPS stimulation increases mechanosensitive ion currents and stiffens cell membranes using patch-clamp electrophysiology techniques. These results indicate that Piezo1 is likely upregulated in the LPS model of cultured astrocytes, thus mechanosensitive responses are increased. Results from these experiments reveal key information about the mechanical properties of Piezo1 and poise Piezo1 as a promising therapeutic for OIH and other neuroinflammatory diseases caused by astrocytic IL-1β release.

Indiana University-Purdue University Indianapolis (IUPUI) / Elaborate vascularization of the retina is crucial for the development and functioning of the eye. The proper patterning of astrocytes is a key event preceding retinal angiogenesis by providing guidance cues for endothelial cells, yet how this is regulated still remains obscure. The dual function of proteoglycans in both extracellular matrix (ECM) composition and cell signal transduction suggests their potential in the regulation of astrocyte migration. The current study demonstrated that non-cell-autonomous regulation by neuroretina cell surface proteoglycan is crucial for PDGF-A regulated astrocyte migration. Ablation of glycosaminoglycan side chains of proteoglycans in neuroretina led to impaired astrocyte migration, incomplete retinal angiogenesis, and hyaloid vessel persistence. This is followed by severe photoreceptor degeneration as a result of reactive gliosis, which cannot be rescued by constitutively activated Kras signaling. Notably, inner limiting membrane (ILM), the basement membrane of the retina, was breached in proteoglycan-deficient retinae prior to the formation of astrocytic network. Herein we propose that cell surface proteoglycans are essential for the initial assembly of ILM, and this cannot be compensated by secreted ECM proteoglycans. In support of this, after removal of ILM in retinal explant by Collagenase digestion, establishment of a new ILM can be achieved by incubation with exogenous laminin-supplemented Matrigel. This basement membrane reconstitution failed, however, in proteoglycan-deficient retinae or in wild type samples digested with a combination of Heparinase and ChABC in addition to Collagenase. Taken together, our study reveals a novel function of neuroretinal cell surface proteoglycans in the initial assembly of basement membrane which subsequently serves as a permissive substratum necessary for astrocyte migration.

Recent epidemiological data have shown that metabolic disease is known to increase the propensity for developing cognitive decline and dementia, particularly Alzheimer’s disease (AD). While this interaction is not completely understood, clinical studies suggest that both hyper- and hypoinsulinemia are associated with an increased risk for developing AD. Indeed, insulin signaling is altered in post-mortem brain tissue from AD patients and insulin and treatments known to enhance insulin signaling, can improve cognitive function. Furthermore, clinical evidence has shown that AD patients and mouse models of AD often display alterations in peripheral metabolism. Since insulin is primarily derived from the periphery it is likely that peripheral alterations can lead to alterations in central nervous system (CNS) insulin signaling and that these changes contribute to cognitive decline. Recent results from our laboratory have shown that in both the APP/PS1 and 3xTg-AD mouse models of AD, peripheral metabolic alterations exist at an early age. Specifically, 3xTg-AD mice demonstrate impaired glucose tolerance at 1 month of age associated with a decrease in insulin and insulin secretion in response to a glucose challenge. This led to the hypothesis that insulin signaling in the CNS would be decreased as a result of decreased peripheral insulin and insulin transport into the CNS. Indeed, insulin signaling through the PI3K/AKT signaling pathway, but not the MAPK/ERK pathway, was decreased in the hippocampus of old, but not young, 3xTg-AD mice. PI3K/AKT signaling can affect several downstream molecules including glycogen synthase kinase 3 (GSK3), glucose transporters (GLUTs), and ATP dependent potassium (KATP) channels. We first examined GSK3 and pTau and found that both GSK3β and pTau were increased in aged 3xTg-AD mice. Next we looked at the translocation of GLUT3 and GLUT4 since both are found in the hippocampus and have recently been shown to be insulin sensitive. Our results showed that GLUT3 translocation, but not GLUT4, was decreased in the hippocampus of aged 3xTg-AD mice. Finally, since KATP channels are found in intracellular organelles as well as in the plasma membrane we examined crude plasma membrane and total fractions of KATP channel subunits Kir6.1 and Kir6.2 and found that the plasma membrane fraction of Kir6.2 was significantly increased. To assess how these changes corresponded with the time course of pathology and cognitive deficits we additionally looked at these changes in 6-8 month and 14-16 month animals. Interestingly, though peripheral insulin was decreased early on, changes in CNS PI3K/AKT insulin signaling did not occur until 18-20 months of age. Changes in GSK3β (but not pTau) and GLUT3 were consistent with this time point suggesting that they were potentially due to the decrease in PI3K/AKT signaling. Since these changes were not consistent with a decrease in peripheral insulin levels it suggests that another factor must be at play. One such factor is inflammation. The AD brain is characterized by inflammation and inflammatory compounds are known to block insulin signaling. KATP channels are not only insulin sensitive but have been shown to play a role in cognition, AD and epilepsy. Thus, to follow up the studies on KATP channels we used immunohistochemistry (IHC), to examine regional and cell specific changes. To our surprise we found that Kir6.2, a subunit typically found primarily in neurons, was present in reactive astrocytes. This finding was further examined in human AD tissue and a similar change was seen. Astrocytes become reactive during damage or under inflammatory conditions, such as AD, diabetes, traumatic brain injury (TBI), epilepsy and in normal aging. When they become reactive both gene expression and functions can change. Since reactive astrocytes and inflammation are a common finding among many neuropathological changes we looked at another neuropathological condition with several similarities to AD, epilepsy. These studies revealed that epileptic mice displayed a similar change in Kir6.2 in reactive astrocytes. Since both conditions are characterized by inflammation we next hypothesized that chronic peripheral inflammation induced by LPS would be enough to drive this change. These studies revealed that while 1 day of LPS treatment was not enough to induce a change in astrogliosis and Kir6.2 expression, three days caused a significant increase in Kir6.2 in reactive astrocytes. This suggests that an increase in Kir6.2 in reactive astrocytes could contribute to the difference in function in these cells and subsequently contribute to altered function in neuropathological disease. Taken together, these studies demonstrate an intricate balance between metabolism and inflammation in the CNS and further suggest that metabolic alterations could be a common link in neuropathological diseases that share similar phenotypic changes, as occurs in AD and epilepsy (i.e. cognitive decline, enhanced seizure susceptibility). Developing a better understanding of metabolism, inflammation, and cortical function/dysfunction could potentially lead to the identification of better treatment options for several neuropathological conditions including AD.

No description available.

Yeung Ho Lam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 93-107). / Abstracts in English and Chinese. / Thesis Committee --- p.i / Abstract --- p.ii / 摘要 --- p.iv / Acknowledgments --- p.v / Table of Contents --- p.vi / List of Abbreviations --- p.x / List of Figures --- p.xiii / List of Tables --- p.xv / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Glucose Transport through the Blood Brain Barrier --- p.1 / Chapter 1.2 --- Roles of Astrocytes in the Brain --- p.4 / Chapter 1.3 --- Glucose Metabolism in Astrocytes --- p.8 / Chapter 1.4 --- Diseases Associated with Reduced Glucose Transport --- p.10 / Chapter 1.5 --- Extracellular Accumulation of Glutamate as a Cause for Epilepsy --- p.13 / Chapter 1.6 --- Regulations of Astrocyte-mediated Glutamate Uptake --- p.17 / Chapter 1.7 --- Aim and Hypothesis of the Project --- p.22 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.24 / Chapter 2.1 --- Materials --- p.24 / Chapter 2.1.1 --- Primary Rat Astrocytes --- p.24 / Chapter 2.1.2 --- Cell Culture Materials --- p.24 / Chapter 2.1.3 --- Chemicals --- p.26 / Chapter 2.1.4 --- Reagents for the Determination of Gene Expressions --- p.26 / Chapter 2.1.5 --- Reagents for the Determination of Protein Expressions --- p.29 / Chapter 2.1.6 --- Reagents for Functional Assays --- p.33 / Chapter 2.1.6.1 --- Reagents for Enzyme-Linked Immunosorbent Assay (ELISA) of IL-6 --- p.33 / Chapter 2.1.6.2 --- Reagents for Glutamate Uptake Assay --- p.33 / Chapter 2.1.6.3 --- Reagents for Extracellular Glutamate Determination Assay --- p.33 / Chapter 2.1.6.4 --- Reagents for Glucose Uptake Assay --- p.34 / Chapter 2.1.6.5 --- Reagents for MTT Assay --- p.34 / Chapter 2.1.6.6 --- Reagents for GFAP Immunostaining --- p.35 / Chapter 2.2 --- Methods --- p.36 / Chapter 2.2.1 --- Preparation of Primary Astrocytes --- p.36 / Chapter 2.2.2 --- Determination of Gene Expressions by Reverse Transcription-Polymersase Chain Reaction (RT-PCR) --- p.37 / Chapter 2.2.3 --- Determination of Protein Expressions by Western Blotting --- p.40 / Chapter 2.2.4 --- ELISA --- p.43 / Chapter 2.2.5 --- Glutamate Uptake Assay --- p.44 / Chapter 2.2.6 --- Extracellular Glutamate Determination Assay --- p.44 / Chapter 2.2.7 --- Glucose Uptake --- p.45 / Chapter 2.2.8 --- MTT Assay --- p.46 / Chapter 2.2.9 --- GFAP Immunostaining --- p.46 / Chapter 2.2.10 --- Band Intensity Quantification --- p.47 / Chapter 2.2.11 --- Statistical Analysis --- p.47 / Chapter CHAPTER 3 --- RESULTS --- p.49 / Chapter 3.1 --- Preparation of Primary Astrocyte Culture --- p.49 / Chapter 3.2 --- Effects of Glucose Deficiency on Astrocyte-mediated Glutamate Uptake --- p.51 / Chapter 3.2.1 --- Effects of Glucose Deficiency on the Expressions of Glutamate Transporters --- p.51 / Chapter 3.2.2 --- Effects of Glucose Deficiency on Glutamate Uptake in Primary Astrocytes --- p.56 / Chapter 3.3 --- Astrocytic Glucose Transport under Glucose Deficiency --- p.61 / Chapter 3.3.1 --- Effects of Glucose Deficiency on the Expressions and Secretion of Inflammatory Cytokines --- p.64 / Chapter 3.3.2 --- Effects of Exogenous Interleukin-6 on Energy Availability in Primary Astrocytes upon Glucose Deficiency --- p.70 / Chapter 3.4 --- Signaling Mechanism Mediating the Astrocytic Responses under Glucose Deficiency --- p.74 / Chapter 3.4.1 --- Effects of Glucose Deficiency on the Expressions of Total and Phosphorylated ERK1/2 in Primary Astrocytes --- p.74 / Chapter CHAPTER 4 --- DISCUSSIONS AND CONCLUSIONS --- p.81 / Chapter 4.1 --- Role of Astrocytes in Preventing Glutamate Excitotoxicity under Glucose Deficiency --- p.81 / Chapter 4.1.1 --- Neonatal Astrocytes as the Cell Model for Chronic Glucose Deficiency --- p.81 / Chapter 4.1.2 --- Effects of Glucose Deficiency on the Expressions of Glutamate Transporters and Glutamate Uptake --- p.83 / Chapter 4.1.3 --- Cytokines: Mediators for Energy Production in Astrocytes --- p.85 / Chapter 4.1.4 --- Summary of the Roles of Astrocyets under Prolonged Glucose Deficiency --- p.88 / Chapter 4.2 --- Establishment of an in vitro GlutlDS model --- p.89 / Chapter 4.3 --- Future Directions of the Project --- p.90 / Chapter 4.4 --- Conclusion --- p.92 / REFERENCES --- p.93 / APPENDIX --- p.108

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Le virus Zika (ZIKV), virus découvert pour la première fois à la fin des années quarante, est un arbovirus émergent récemment arrivé sous le feu des projecteurs à l’occasion d’une pandémie rapide à l’échelle mondiale. Appartenant à la famille des Flaviviridae, ce flavivirus est transmis par les moustiques du genre Aedes. Alors qu’on le croyait relativement peu pathogène, ce virus se révèle être la cause probable d’une vague de complications neurologiques, incluant l’apparition de microcéphalies et de syndromes de Guillain-Barré. De plus, il n’existe à l’heure actuelle ni vaccins ni traitements spécifiques, la lutte contre le virus se résumant largement à la mise en place de mesures de prévention contre la piqûre de moustiques et la lutte anti-vectorielle.Une meilleure connaissance de l’ensemble de la biologie du virus, depuis les modalités d’entrée dans l’organisme, en particulier au niveau cutanée, jusqu’aux mécanismes moléculaires intimes de la réplication du virus s’avère nécessaire. Par des approches moléculaires et cellulaires, nous avons mis en évidence le tropisme du virus, identifié ses récepteurs et déterminé les réponses cellulaires induites par ce dernier. Nos travaux ont également identifié un potentiel mécanisme d’évasion mise en place par le ZIKV. Nous avons également entrepris un travail original sur un mécanisme moléculaire favorisant la pathogénicité des flavivirus. Une meilleure connaissance de ce mécanisme pourrait déboucher sur l’identification de potentiels cibles thérapeutiques. Enfin, le tropisme neuronal avéré du ZIKV nous a amené à travailler sur la réponse immune des astrocytes humain. En effet, les astrocytes forment une population cellulaire très importante dans le système nerveux central qui est fortement impliquée dans les mécanismes de neurogénèse dans le cerveau des fœtus. / The Zika virus (ZIKV) was first isolated from non-human primates the late 1940s. This emerging arbovirus has recently been under the spotlight due to a rapid world pandemic. Belonging to the Flaviviridae family, this flavivirus is transmitted by Aedes’ genus mosquitoes. Historically low pathogenic, a new major concern is the possible association of ZIKV with diverse of neurological complications, including the development of microcephaly and Guillain-Barré syndrome, particularly in newborns of infected mothers. In addition, there is currently no vaccine or specific treatment to cure the disease, so the main preventive measures to fight the spreading of the virus are to prevent mosquitoes’ bites and to plan an effective vector control. A better understanding of the biology of the virus, from the entry in the body, especially at the skin level, to the molecular mechanisms of viral replication, is therefore necessary.Using different molecular and cellular strategies, we investigated the tropism of the virus, identified cell surface receptors and determined the cell’s responses to the infection. Our work also permitted to identify a potential mechanism by which ZIKV evades the host immune system to facilitated his own replication. We also have undertaken original work on a molecular mechanism increasing the pathogenicity of flavivirus. A better knowledge of this mechanism may lead to the identification of potential therapeutic targets. Finally, considering the neuronal tropism of the ZIKV, we studied the immune response of human astrocytes, a very important cell population in the central nervous system, playing a major role in the mechanisms of neurogenesis during the fetus’ brain development.

Les douleurs neuropathiques, caractérisées par une prévalence élevée, résultent de la compression ou lésion de nerfs. Les antidépresseurs figurent parmi les traitements de première intention de cette pathologie, toutefois des besoins médicaux restent insatisfaits par ces molécules. Mon travail de thèse s'est intéressé à la connexine 43 (Cx43) astrocytaire impliquée dans le mécanisme d'action des antidépresseurs ainsi que dans les douleurs neuropathiques. La première partie des travaux, réalisée avec des cultures d'astrocytes corticaux de souris, a permis de préciser le lien entre les antidépresseurs et la Cx43 astrocytaire. Alors que son expression s'est avérée inchangée dans notre modèle, un effet hétérogène des antidépresseurs a été observé sur le couplage intercellulaire des astrocytes. L'ensemble des molécules testées a conduit à l'inhibition de l'ouverture des hémicanaux de Cx43 étudiée dans un contexte inflammatoire, notre étude est la première à rapporter cet effet. De plus, les antidépresseurs prescrits dans les douleurs neuropathiques ont induit l'inhibition du couplage et/ou des hémicanaux. La seconde partie de mes travaux a porté sur la combinaison entre l'amitriptyline et la méfloquine. Ceci repose sur les approches combinatoires proposées par Theranexus, start-up avec qui la thèse a été réalisée. Les deux molécules ont présenté une synergie d'effet sur la réduction du couplage astrocytaire in vitro corrélée a une potentialisation de l'action anti-hyperalgésique de l'amitriptyline in vivo, chez le rat lésé au niveau du nerf sciatique. Ces résultats valident l'implication de la Cx43 astrocytaire dans la réponse antinociceptive des antidépresseurs. / Neuropathic pain, characterised by a marked prevalence, is the consequence of nerve compression or lesion. Antidepressants represent the main treatments of this disease, however, medical needs remain mostly unsatisfied by these molecules. In order to improve this therapeutical approach, my thesis work has focussed on astroglial connexin 43 (Cx43) that has recently been involved in the mechanism of action of antidepressant as well as in neuropathic pain. The first part of my work, performed in primary cultures of mouse cortical astrocytes, has allowed to reinforce the link between antidepressants and Cx43 in astrocytes. Whereas its expression was unchanged in our model, an heterogeneous effect of antidepressants was observed on the intercellular communication of astrocytes. Furthermore, all tested molecules led to the inhibition of Cx43 hemichannel activity in an inflammatory context, our study is the first to report such effect. Interestingly, antidepressants prescribed in neuropathic pain induced inhibition of coupling and/or hemichannels. The second part of my work concerned the combination between amitriptyline and mefloquine. This is based on combinatorial approaches proposed by Theranexus, a start-up partner in this project. The association of the two molecules presented a synergy on astroglial coupling reduction in vitro correlated to a potentiation of the anti-hyperalgesic effect of amitriptyline in vivo, in rats with injured sciatic nerve. These results confirm the implication of the astroglial Cx43 in the antinociceptive response to antidepressants.

Le tabagisme actif par la mère expose le fœtus en développement à des agents qui peuvent traverser la barrière placentaire et interférer avec les fonctions placentaires. Un large éventail de fonctions immunologiques, pourrait être compromises. Dans cette étude, nous avons évalué l'effet de l'extrait de la fumée de cigarette (CSE) sur les macrophages isolés à partir de placentas humains (pMφs), qui sont les principaux partenaires de l'immunité de fœto-maternelle innée. J’ai pu montrer que le CSE inhibe la formation des cellules géantes multinucléées (MGC). Cette propriété du CSE est spécifique aux macrophages car la fusion des macrophages dérivés des monocytes est inhibée lors de la formation de granulomes in vitro. J’ai également étudié l'absorption de particules et la production de cytokines par pMφs exposés au CSE. Le CSE a inhibé l'absorption des particules de zymosan, mais pas celle du zymosan opsonisé, ce qui suggère qu’il interfère avec les récepteurs phagocytaires et non phagocytaires. Le CSE augmente la libération de TNF et d'IL-33, et une diminue celle de l'IL-10, ce qui montre que l'équilibre entre les cytokines est affecté par le CSE. En outre, l’expression des métalloprotéinases telles que les MMP-1, MMP-10 et MMP-12, connues pour être impliquées dans le remodelage des tissus et la fusion des macrophages est dérégulée. Enfin, j’ai montré que la nicotine, l'un des principaux composés de tabac, n'a pas affecté les propriétés fonctionnelles des pMφs. / Active smoking by the mother exposes the developing fetus to agents that can cross the placental barrier and interfere with placental functions. A wide range of immunological functions, including innate and adaptive immune responses, might be impaired. In this study, we assessed the effect of cigarette smoke extract (CSE) on macrophages isolated from human placentas (pMφs), which are major partners of innate feto-maternal immunity. I showed that CSE significantly inhibited the formation of multinucleated giant cells (MGCs). This property of CSE is specific to macrophages because the fusion of monocyte-derived macrophages is inhibited during the in vitro formation of granulomas. I also investigated particle uptake and cytokine production by pMφs exposed to CSE. CSE inhibited the uptake of zymosan, but not that of opsonized zymosan, suggesting that it interferes with phagocytic receptors, not with the phagocytic machinery of pMφs. CSE increased the release of Tumor Necrosis Factor and interleukin-33, and decreased that of interleukin-10, demonstrating that the balance between inflammatory and anti-inflammatory cytokines is affected by CSE. Furthermore, CSE enhanced the expression of metalloproteinase (MMPs) genes such as MMP-1, MMP-10 and MMP-12, known to be involved in tissue remodeling including macrophage fusion. Finally, I showed that nicotine, one of the major compounds of tobacco, did not affect the functional properties of pMφs.