Background: Current recommended treatment options for primary headache disorders are suboptimal in that effective pain relief is untimely and associated with side effects. Objective: The primary aim of this thesis was to evaluate the effectiveness and attitudes towards an alternative treatment option, the sphenopalatine ganglion (SPG) block which belongs to a class of procedures known as peripheral nerve blocks (PNBs). Methods: We conducted a systematic review and meta-analysis studying the effectiveness of PNBs for the treatment of primary headache disorders. We also conducted a national postal survey among Canadian emergency physicians to identify current practice patterns and perspectives on PNBs. Results: We found PNBs are effective for rapid pain relief compared to placebo at 15 minutes (MD = -1.17; 95% CI: -1.82 to -0.51) and 30 minutes (MD = -0.99; 95% CI: -1.66 to -0.32). Only 55.6% of physicians have experience with PNBs (95% CI: 0.48 to 0.65) and we discovered the SPG block is the least commonly practiced type of PNB; the majority of physicians believe these procedures are safe (85%) and effective (55.1%). A future trial is needed to compare the SPG block against standard treatment. Conclusion: The SPG block may be used as an adjunct therapy for faster effective pain relief. Current physicians would consider PNBs as a first-line alternative given evidence from a future trial. A future trial is needed to compare the SPG block against standard treatment.

No description available.

Les modifications post-traductionnelles (MPTs) permettent un haut degré de régulation de l'expression des gènes en générant une diversité fonctionnelle au niveau du protéome. Dans le système nerveux, les MPTs régulent entre autres des facteurs de transcription permettant une adaptation rapide à un microenvironnement dynamique. Dans ce contexte, je me suis concentrée sur l’étude des Glycogène Synthase Kinases 3 (GSK3s). Elles sont au centre de la régulation de nombreuses voies de signalisation et contrôlent la stabilité de multiples cibles par phosphorylation. Au cours du développement du cerveau, les kinases GSK3 contrôlent la balance entre la prolifération et la différenciation. La dérégulation de l'activité des kinases GSK3 a un rôle clé dans les maladies neurodégénératives du cerveau. En revanche, le rôle important de ces kinases au cours du développement rétinien ainsi que dans les maladies neurodégénératives rétiniennes reste une question ouverte.L'objectif de ma thèse était d'étudier le rôle de ces kinases au cours du développement et de l'homéostasie rétinienne. J’ai montré que l'absence totale de Gsk3α et de Gsk3β très tôt au cours du développement rétinien entraîne une microphtalmie chez l'adulte. Les deux kinases jouent des rôles redondants puisque l'expression d'un seul allèle Gsk3 est suffisante pour prévenir le phénotype de microphtalmie. Cependant, une analyse phénotypique approfondie dans ce contexte génétique (un seul allèle Gsk3) a révélé une forte augmentation du nombre de cellules ganglionnaires déplacées (dRGCs) dans la couche nucléaire interne, associée à une modification des projections axonales des cellules ganglionnaires dans le cerveau par rapport aux contrôles. Dans l’ensemble, ces données suggèrent que les kinases GSK3s sont essentielles au maintien des progéniteurs rétiniens et sont impliquées dans la genèse des dRGCs. Compte tenu du très faible nombre de dRGCs en conditions normales, la fonction de ces cellules a été très peu étudiée à ce jour. Le modèle génétique que j’ai développé offre par conséquent un modèle de choix pour étudier l’ontogenèse et la fonction de ces cellules.Mes travaux de thèse se sont ensuite concentrés sur le rôle de GSK3 dans les photorécepteurs. En effet, des défauts de développement ou leur mort est l’une des principales causes de dégénérescence rétiniennes. Afin de mieux comprendre la fonction de ces kinases dans la maintenance des photorécepteurs, j'ai donc utilisé des souris invalidées de manière conditionnelle pour Gsk3α et Gsk3β spécifiquement dans les précurseurs des photorécepteurs. L’absence de GSK3 conduit à une altération de la maturation et de la fonction des photorécepteurs, suivie de leur dégénérescence. J’ai alors combiné des analyses transcriptomiques et des approches in vitro pour élucider les mécanismes sous-jacents. Mes données m’ont conduit à proposer un modèle selon lequel l’absence de GSK3 dans les photorécepteurs conduit à des défauts de phosphorylation de NRL (facteur de transcription nécessaire au développement des photorécepteurs de type bâtonnet), augmentant sa stabilité. Cette dérégulation post-traductionnelle conduit à la diminution d’expression d'un sous-ensemble de gènes cibles de NRL, co-régulés par CRX, et impliqués dans le développement et l'homéostasie des photorécepteurs. Cette dérégulation conduirait alors à la dégénérescence des photorécepteurs observée dans les mutants GSK3. Ce travail suggère donc que GSK3 joue un rôle essentiel dans la régulation de NRL pour contrôler la maturation et l'homéostasie des photorécepteurs. De telles données suggèrent également que ce mécanisme de régulation pourrait être déficient chez les patients atteints de rétinites pigmentaires dues à des mutations de NRL empêchant sa phosphorylation par GSK3. / Post-translational modifications (PTMs) allow a higher degree of regulation for the control of gene expression by generating functional diversity at the proteome level. In the central nervous system, PTMs regulate stability or activity of transcription factors allowing a rapid response to external signals and a quick adaptation to a dynamic cellular microenvironment. In this context, I focused on the ubiquitously expressed and highly conserved Glycogen Synthase Kinases 3 (GSK3s). They are at the crossroad of multifunctional signalling pathways. During mammalian brain development, GSK3 kinases control the balance between proliferation and differentiation. Deregulation of GSK3 kinases activity has also a key role in neurodegenerative diseases by causing the accumulation/aggregations of proteins causing neuronal cell death. Drugs targeting GSK3s hold a lot of promises to treat such diseases. Whether these kinases are also important during retinal development and involved in retinal diseases remains an open question. Several studies suggest the importance of regulating GSK3 function in photoreceptor under pathological conditions. Therefore, the main objective of my PhD was to investigate the role of these kinases during photoreceptor development and homeostasis. To better understand the role of these two kinases during retinal development and to highlight potential differences with the developing brain, we also investigated their function in the control of the balance between proliferation and differentiation of retinal progenitors. To achieve my work, I used conditional knockout mice for Gsk3α and Gsk3β specifically deleted either in photoreceptor precursors or in retinal progenitors during early development. The lack of GSK3 kinases in photoreceptor precursors led to impaired photoreceptor maturation and function followed by their degeneration. Transcriptomic analysis (RNAseq) 6, 10 and 14 days postnatally prior degeneration revealed several genes downregulated belonging to biological processes involved in eye development and visual functions. Among them, the expression of the transcription factor Nrl that is required for rod photoreceptor development was decreased. Astonishingly, NRL expression was highly increased at protein level. By in vitro approaches, I demonstrated that GSK3-dependent phosphorylation regulates NRL protein stability. Despite such increase, a large number of NRL target genes were downregulated leading to impaired photoreceptor maturation and function. Surprisingly, a vast majority of these downregulated genes were also target genes for CRX, another transcription factor working in synergy with NRL. This work demonstrates that PTMs of NRL play a critical role in fine tuning the expression of a subset of genes involved photoreceptor development and homeostasis. Such findings could allow the development of innovative therapeutic strategies for retinal dystrophies. The functional characterisation of GSK3 in the course of retinal development by invalidating both Gsk3α and Gsk3β in retinal progenitors early during development revealed their requirement for controlling cell cycle exit and neuronal differentiation. Indeed, the complete lack of Gsk3α and Gsk3β led to microphtalmia in adults. Interestingly, the expression of only one Gsk3 allele was enough to rescue the phenotype. However, further analysis revealed a large number of displaced ganglion cells in the inner nuclear layer. The function of these cells remains to be determined, but their timing of production corresponds to other ganglion cells. Strikingly, these displaced ganglion cells project in distinct brain regions than normal ganglion cells. Therefore, our work could provide the first step toward determining the function of the displaced ganglion cells, which appear at low number in wildtype but whose function remains to be clarified.

Les cellules de Schwann (CS) sont les cellules gliales myélinisantes du Système Nerveux Périphérique (SNP). Il existe une communication étroite entre ces cellules et les axones auxquels elles s’associent et ce dès les stades les plus précoces de leur développement. Elles migrent tout en se divisant le long des axones; cette division migratoire est suivie d’une deuxième division post-migratoire dans le but d’établir un ratio 1:1 avec les axones pour ensuite les myéliniser. Ce travail vise à analyser, in vivo, le comportement des CS chez le poisson zèbre au cours de leurs divisions.Nous avons remarqué que les CS se divisent parallèlement aux axones le long du nerf de la Ligne Latérale Postérieure (PLL). En analysant les deux mutants has et nok, nous avons montré que les gènes de polarité apicale aPKC et pals1 ne sont pas requis pour la migration et la division des CS, ni pour leur capacité à myéliniser. Nous avons mis en évidence, en analysant le mutant cassiopeia qui présente des défauts d’organisation du fuseau mitotique et en utilisant l’agent pharmacologique le nocodazole, que l’assemblage du fuseau mitotique au cours de la division des CS est essentiel pour la myélinisation.En parallèle, nous avons analysé le rôle du gène rgs4 (regulator of G-protein Signaling 4) dans le développement du SNP chez le poisson zèbre. Nous avons généré un mutant stable rgs4 par la technique CRISPR/Cas9 et montré un rôle de ce gène dans le développement du ganglion de la PLL et des motoneurones, et ce en agissant en amont de la voie PI3K/Akt/mTOR.Contrairement à l’inhibition pharmacologique qui suggère un rôle de rgs4 dans la myélinisation périphérique, le mutant ne présente pas de défauts de myéline. / Schwann cells (SCs) are the myelinating glial cells of the Peripheral Nervous System (PNS). They derive from neural crest cells during development, then migrate and divide along the axons of the peripheral nerves. This migratory division is followed by a post-migratory division in order to radially sort the axons in a 1:1 ratio and wrap them with a myelin sheath. This work provides an analysis of the polarity of SC divisions, in vivo, in intact zebrafish embryos.We showed that SCs divide parallel to the axons along the Posterior Lateral Line nerve (PLL). By analyzing the two mutants has and nok, we revealed that the apical polarity genes aPKC and pals1, are neither required for the migration and division of SCs, nor for their capacity to myelinate. By studying the cassiopeia mutant that shows defects in mitotic spindle, we revealed that the assembly of the mitotic spindle is essential for SC myelination.We have also analysed the role of rgs4 (regulator of G-protein Signaling 4) in PNS development. We generated a stable rgs4 mutant using the CRISPR/Cas9 technology. We showed that rgs4 plays an essentiel role in PLLg and motoneurons development by acting upstream of PI3K/Akt/mTOR pathway. Pharmacological analysis suggested a role for rgs4 in peripheral myelination, however, the rgs4 mutant do not show any myelin defects.

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No description available.

1. The effects of external H-7, a potent protein kinase inhibitor, on the responses mediated by γ-aminobutyric acid A type (GAGA(A))-, nicotinic acetylcholine (nicotinic ACh)-, ionotropic 5-hydroxytryptamine (5-HT3)-, adenosine 5'-triphosphate (ATP)-, N-methyl-D-aspartate (NMDA)- and kainate (KA)-receptors were studied in freshly dissociated rat dorsal root ganglion neurone by use of whole cell patch-clamp technique. 2. External H-7 (1-1000 μM) produced a reversible, dose-dependent inhibition of whole cell currents activated by GABA, ACh and 5-HT. 3. Whole-cell currents evoked by ATP, 2-methylthio-ATP, NMDA and KA were sensitive to external H-7. 4. External H-7 shifted the dose-response curve of GABA-activated currents downward without changing the EC50 significantly (from 15.0 ± 4.0 μM to 18.0 ± 5.0 μM). The maximum response to GABA was depressed by 34.0 ± 5.3%. This inhibitory action of H-7 was voltage-independent. 5. Intracellular application of H-7 (20 μM), cyclic AMP (1 mM) and BAPTA (10 mM) could not reverse the H-7 inhibition of GABA-activated currents. 6. The results suggest that external H-7 selectively and allosterically modulates the functions of GABA(A)-, nicotine ACh- and 5-HT3 receptors via a common conserved site in the external domain of these receptors.

<p>Glaucoma is a leading cause of blindness characterized by the progressive loss of retinal ganglion cells (RGCs), essentially severing the connection between the eye and the brain. Among many underlying causes of the disease, mutations in the Optineurin (OPTN) gene result in severe RGC neurodegeneration in the absence of elevated intraocular pressure, providing a novel opportunity to study molecular mechanisms that lead to RGC neurodegeneration associated with glaucoma. Efforts of this study establishing a human pluripotent stem cell (hPSC)-derived in vitro disease model by inserting OPTN(E50K) mutation via CRISPR/Cas9 genome editing and investigate the cellular mechanisms of RGC neurodegeneration associated with glaucoma. OPTN(E50K) RGCs revealed neurodegeneration phenotypes, including downregulation of RGCs transcription factors, neurite retraction, and hyperexcitability, suggesting that OPTN(E50K) RGCs can serve as an appropriate disease model to study glaucoma-associated neurodegeneration. Since OPTN serves a primary role as an autophagy receptor, we further hypothesized that the OPTN(E50K) mutation disrupts autophagy in RGCs, and modulation of autophagy by mammalian target of rapamycin (mTOR)-independent pathways can preserve RGC phenotypes by maintaining mTOR signaling. OPTN(E50K) RGCs exhibited a higher number of OPTN puncta along with an overall reduced expression of OPTN protein, indicating a gain of toxic protein accumulation or loss of protein function. Furthermore, OPTN(E50K) RGCs revealed an accumulation of the autophagosome protein LC3 in a punctal manner as well as increased expression of lysosomal proteins, suggesting a disruption of degradation pathway in autophagosome and lysosome fusion. As mTOR complex 1 (mTORC1) signaling serves as a negative regulator of autophagy, a downregulation of mTORC1 signaling via activation of stress sensor adenosine monophosphate-activated protein kinase (AMPK) was observed as a possible compensatory mechanism for autophagy deficits in OPTN(E50K) RGCs. Pharmacological inhibition of mTOR in wild-type hRGCs resulted in similar disease-related phenotypes, while preservation of the mTOR pathway in OPTN(E50K) RGCs by treatment with the mTOR-independent autophagy modulator trehalose cleared OPTN accumulated puncta, preserving mTORC1 signaling, as well as rescuing neurodegenerative phenotypes. To further validate these associations in an animal model, the microbead occlusion mouse model was established by injection of magnetic microbeads in the anterior chamber to block aqueous outflow resulting ocular hypertension. In agreement with our findings in hRGCs, a decrease in mTOR signaling associated with an increase in the expression of autophagy-associated proteins was observed in RGCs in the microbead occlusion model. Additionally, these disease-related phenotypes were observed specifically within RGCs but not cortical neurons with an underlying OPTN(E50K) mutation, demonstrating that autophagy represents an essential pathway in RGCs to maintain homeostasis, and selective disrupt of autophagy in RGCs leads to neurodegeneration. Taken together, the results of this study highlight an essential balance between autophagy and mTORC1 signaling that is essential for the homeostasis of RGCs, while disruption to these signaling pathways contributes to neurodegenerative features in glaucoma. These results also demonstrated the ability to pharmacologically intervene to experimentally manipulate these pathways and rescue neurodegenerative phenotypes, providing a potential therapeutic target to prevent glaucoma-associated neurodegeneration. </p>

Indiana University-Purdue University at Indianapolis (IUPUI) / Human pluripotent stem cells (hPSCs) are an attractive tool for the study of different neurodegenerative diseases due to their potential to form any cell type of the body. Due to their versatility and self-renewal capacity, they have different applications such as disease modeling, high throughput drug screening and transplantation. Different animal models have helped answer broader questions related to the physiological functioning of various pathways and the phenotypic effects of a particular neurodegenerative disease. However, due to the lack of success recapitulating some targets identified from animal models into successful clinical trials, there is a need for a direct translational disease model. Since their advent, hPSCs have helped understand various disease effectors and underlying mechanisms using genetic engineering techniques, omics studies and reductionist approaches for the recognition of candidate molecules or pathways required to answer questions related to neurodevelopment, neurodegeneration and neuroregeneration. Due to the simplified approach that iPSC models can provide, some in vitro approaches are being developed using microphysiological systems (MPS) that could answer complex physiological questions. MPS encompass all the different in vitro systems that could help better mimic certain physiological systems that tend to not be mimicked by in vivo models. In this dissertation, efforts have been directed to disease model as well as to understand the intrinsic as well as extrinsic cues using two different MPS. First, we have used hPSCs with Alzheimer’s disease (AD)-related mutations to differentiate into retinal organoids and identify AD related phenotypes for future studies to identify retinal AD biomarkers. Using 5 month old retinal organoids from AD cell lines as well as controls, we could identify retinal AD phenotypes such as an increase in Aβ42:Aβ40 ratio along with increase in pTau:Tau. Nanostring analyses also helped in identification of potential target genes that are modulated in retinal AD that were related to synaptic dysfunction. Thus, using retinal organoids for the identification of retinal AD phenotypes could help delve deeper into the identification of future potential biomarkers in the retina of AD patients, with the potential to serve as a means for early identification and intervention for patients. The next MPS we used to serve to explore non-cell autonomous effects associated with glaucoma to explore the neurovascular unit. Previous studies have demonstrated the degeneration of RGCs in glaucoma due to a point mutation OPTN(E50K) that leads to the degeneration of RGCs both at morphological and functional levels. Thus, using the previous studies as a basis, we wanted to further unravel the impact of this mutation using the different cell types of the neurovascular unit such as endothelial cells, astrocytes and RGCs. Interestingly, we observed the barrier properties being impacted by the mutation present in both RGCs and astrocytes demonstrated through TEER, permeability and transcellular transport changes. We also identified a potential factor TGFβ2 that was observed to be overproduced by the OPTN E50K astrocytes to demonstrate similar effects with the exogenous addition of TGFβ2 on the barrier. Furthermore, the inhibition of TGFβ2 helped rescue some of the barrier dysfunction phenotypes. Thus, TGFβ2 inhibition can be used as a potential candidate that can be used to further study its impact in in vivo models and how that can be used in translational applications. Thus, MPS systems have a lot of applications that can help answer different physiologically relevant questions that are hard to approach using in vivo models and the further development of these systems to accentuate the aspects of neural development and how it goes awry in different neurodegenerative diseases.

No description available.