The neuronal ceroid lipofuscinoses (NCLs) are a family of neurodegenerative diseases predominantly affecting infants and children, which in some cases can present into adulthood. There are fourteen genes comprising the 13 known subtypes of NCLs (CLN1-CLN8, CLN10-CLN14; CLN9 has been reclassified as CLN4). The NCL diseases share common molecular and clinical features, including cellular accumulation of autofluorescent storage material, characteristic histological findings (curvilinear inclusions, fingerprint profiles, and granular osmophilic deposits), markedly low brain weight, seizures, blindness, motor dysfunction and behavioral disabilities. Though the functions of the CLN proteins are not fully understood, they are mainly localized to the lysosomal compartment and autophagic pathway. Previous works have focused on understanding the individual functions of the CLN proteins. However, there is little research examining the interactions between CLN proteins and their involvement in neurogenesis. The CLN proteins also show involvement in various other signaling pathways, notably the mTOR and p53 pathways, and may therefore have implication as important signaling molecules during development and aging. In this thesis, I outline a variety of interactions between CLN proteins, as well as their role in lysosome formation and autophagy. I further examine the involvement of these proteins in lysosomes of microglia, and potential functions of microglia during neurogenesis in childhood and adulthood. I hypothesize that the CLN proteins are likely involved in a common pathway which is highly regulated during neurogenesis through microglial release of pro-inflammatory molecules. Though these diseases are incurable, enzyme replacement shows promise as a treatment for NCL; cerliponase alpha (BioMarin Pharmaceuticals) is the first and only FDA-approved enzyme replacement treatment for CLN2 disease. Future in-depth investigation of protein-protein interactions as well as their involvement in signaling pathways during development is necessary in order to find a cure for these devastating diseases.

The aim of this study is to describe hospice caregivers’ perceptions and practices in the management of dysphagia in neurodegenerative disorders. In South Africa, hospices provide support and care for people with neurodegenerative disorders and have been offering palliative care to patients and their families facing life-threatening illnesses since 1989. Detailing the management practices of hospice caregivers may assist in determining whether there is a need for the services of a speech-language therapist in the transdisciplinary model. A non-experimental, descriptive survey research design was selected for the purpose of this study as it allowed the researcher to describe the characteristics of a large number of respondents. Information was gathered by means of mail-distributed, self-administered questionnaires. The collected data was descriptively analysed and graphs and figures were used to summarise and display the frequency distribution and associations within the data. It was evident from the findings that dysphagic patients with neurodegenerative disorders form part of the hospice caregivers’ caseloads. However, there appears to be limited specialist involvement in the management of neurodegenerative dysphagia within the hospice setting. Furthermore, caregivers’ perceptions of the subtle symptoms of dysphagia, the positions that facilitate safe swallowing and the consistencies that are most easily swallowed by people with dysphagia were found to be inadequate. The results obtained have numerous significant clinical and theoretical implications regarding current dysphagia management in the South African hospice setting. Recommendations are made to hospices, speech-language therapists and future researchers. / Dissertation (MCommunication Pathology)--University of Pretoria, 2009. / Speech-Language Pathology and Audiology / unrestricted

Philosophiae Doctor - PhD / Neurodegenerative disorders are characterised by progressive loss of the brain’s physiological functions as a result of gradual degeneration of neurons in the central nervous system. Even though they are classified as diseases of the elderly, occurrence earlier in life is possible, but that would suggest the influence of genetic and/or environmental factors. Due to the continuous rise in modernisation and industrialisation over the years, there has been an increase in incidence and prevalence of neurodegenerative disorders. With the advances in technology and life expectancy, the rates of the common forms (Alzheimer’s disease and Parkinson’s disease), are expected to increase exponentially by 2050. Unfortunately, there is still no clinically approved treatment or therapy to slow down or halt the degenerative process as most registered drugs only offer symptomatic relief. Confounding this issue is the lack of definite mechanism of neurodegeneration, which is still poorly defined and not completely understood. Nonetheless, the pathology of most neurodegenerative disorders is believed to be a combination of interrelated processes that eventually leads to neuronal cell death. Among the postulated processes, the impact of excitotoxicity mediated by NMDA receptor over-activation is prominent and it is implicated in virtually all neurodegenerative disorders. With this basic insight, it is believed that molecules capable of inhibiting NMDA receptors and associated calcium channels, without affecting the normal physiological functions of the brain, could potentially serve as good neuroprotective drugs. Competitive and uncompetitive blockers (MK-801 and ketamine) have been explored, but none were clinically accepted due to undesirable side effects such as hallucinations, sedation and depression. However, NGP1-01, a polycyclic cage molecule, has been shown to be neuroprotective through modulation of NMDA receptors and voltage gated calcium channels and attenuation of MPP+ -induced toxicity. A similar approach could be useful in the design and development of new neuroprotective drugs. The aim of this study was to synthesise a series of open and rearranged cage-like molecules and explore their neuroprotective potential in neuroblastoma SH-SY5Y cells. The proposed structures, with norbornane scaffolds that contained different moieties, were designed to structurally resemble NGP1-01 and MK-801. Once synthesised, the compounds were purified and characterised, and were evaluated for their biological activities. Compounds were first screened for cytotoxicity at different concentrations. Thereafter, they were evaluated for neuroprotective effects against MPP+ -induced excitotoxicity and for calcium flux modulatory effects on NMDA receptor and voltage gated calcium channels. The norbornane derivatives were synthesised and characterised, and all final products were afforded in sufficient yields. All compounds with the exception of two compounds displayed good cytotoxic profiles towards the SH-SY5Y neuroblastoma cells at 10 µM, 50 µM and 100 µM concentrations as they demonstrated percentage cell viabilities close to 100% (control treated cells). Only two compounds showed percentage cell viability of 51% and 59% at 100 µM. Utilising the same cell line, all compounds, tested at 10 µM, attenuated MPP+ -induced toxicity after 24 hours of exposure to a neurotoxin. This was evident in the 23% to 53% enhancement (significant with p < 0.05) in cell viability when compared to the MPP+ only treated cells. In comparison to known NMDA receptor and/or voltage gated calcium channel blockers (MK-801, NGP1-01 or nimodipine), the synthesised compounds demonstrated mono or dual inhibition of calcium channels as they effectively attenuated calcium influx by blocking NMDA receptors and/or voltage gated calcium channels expressed in neuroblastoma SHSY5Y cells. This group of compounds were found to be more potent NMDA receptor inhibitors, probably due to similarities with MK-801 and memantine, than voltage gated calcium channel inhibitors. All compounds demonstrated moderate to good calcium inhibitory effects at NMDA receptors in the range of 23% to 70% while a selected few displayed very little or no activity at the voltage gated calcium channels. In conclusion, 27 compounds with norbornane scaffolds were successfully synthesised and evaluated for cytotoxicity and neuroprotection. The abilities of the synthesised compounds to protect neurons from the neurotoxin MPP+ and reduce calcium flux into neuronal cells were successfully demonstrated. These characteristics are essential in neuroprotection as they may prove significant in halting or slowing down the disease progression. The compounds showing a good cytotoxicity profile, neuroprotective effects and ability to reduce calcium overload, could potentially act as neuroprotective agents with good safety profiles or contribute as lead structures to the development and design of structurally related molecules that could clinically benefit people with neurodegenerative disorders.

No description available.

Parkinson’s Disease (PD) is a neurodegenerative disorder, within this categoryof diseases it is among the most prevalent worldwide. The etiology of PD isbased in progressive deterioration of neural tissue in the basal ganglia (neuronalnuclei located at the base of the cerebrum) and their related structures. Current research is focusing on treatment approaches to either enhance or replaceexisting pharmaceutical treatment approaches, such as dopamine replacementtherapy. In this project, the focus was on finding correlates between movementdata and neurological signals to provide insight into potential biomarkers forcomplex motor symptoms of PD. This will in turn provide a starting point forspecifically targeted closed-loop neural stimulation that alleviates these symptoms. Although the data available at the time of this thesis did not providesufficient insight to derive a conclusion on the neural correlates, a pipeline wasdeveloped, which analyzes and synchronizes kinematic and neural data and willenable further exploration as additional data is obtained.

There has in recent years been interdisciplinary research on utilizing machine learning for detecting and classifying neurodegenerative disorders with the sole goal of outperforming state-of-the-art models in terms of metrics such as accuracy, specificity, and sensitivity. Specifically, these studies have been conducted using existing networks on ”novel” methods of pre-processing data or by developing new convolutional neural networks. As of now, no work has looked into how different normalization techniques affect a deep or shallow convolutional neural network in terms of numerical stability, its performance, explainability, and interpretability. This work delves into what normalization technique is most suitable for deep and shallow convolutional neural networks. Two baselines were created, one shallow and one deep, and applied eight different normalization techniques to these model architectures. Conclusions were drawn based on our analysis of numerical stability, performance (metrics), and methods of Explainable Artificial Intelligence. Our findings indicate that normalization techniques affect models differently regarding the mentioned aspects of our analysis, especially numerical stability and explainability. Moreover, we show that there should indeed be a preference to select one method over the other in future studies of this interdisciplinary field.

La mucopolysaccharidose de type VII (MPS VII) est une maladie génétique de surcharge lysosomale provoquée par une déficience en β-D-glucuronidase (β-glu). β-glu est impliquée dans la cascade de dégradation et de recyclage des glycosaminoglycannes (GAGs). Sa déficience provoque une accumulation vésiculaire de GAGs non dégradés engendrant in fine la mort cellulaire. Notre but est de développer et de tester la pertinence des vecteurs adénoviraux canins helper-dépendant (HD-CAV-2) pour traiter la neurodégénération provoquée par la MPS VII dans le cadre d'une thérapie génique dans le SNC de chien. Parce que les vecteurs CAV-2 transduisent préférentiellement les neurones et qu'ils sont transportés de manière rétrograde le long des axones, leurs distributions dans tout le SNC permettraient de délivrer largement la β-glu dans tout le parenchyme cérébral. Nous avons alors étudié la sureté, la durée d'expression, l'efficacité et la possible réversion du phénotype après injections dans le SNC de chiens MPS VII d'un HD-CAV-2 exprimant le gène humain de la GUSB : HD-RIGIE. Des études préliminaires ont montré la faisabilité du transfert des vecteurs HD-CAV-2 dans le SNC, qu'ils induisaient une réponse immunitaire minimale et qu'ils transduisaient préférentiellement, efficacement et largement les neurones.Nous avons produit un HD-RIGIE de qualité pour les injections intracérébrales et nous avons analysé son efficacité sur l'accumulation de GAGs non dégradés. Les injections de HD-RIGIE montrent une augmentation générale de l'activité β-glu dans tout le SNC des chiens MPS VII (sites d'injections et structures éloignées comme le cortex) et ce 1 mois ou 4 mois après les injections. L'analyse de la GFP confirme une distribution globale de HD-RIGIE dans le SNC d'animaux de grande taille. De plus, grâce aux propriétés intrinsèques de la β-glu (transport rétrograde et phénomène de libération/recapture), nous avons observé une diminution générale de l'accumulation vésiculaire neuronale des GAGs non dégradés dans tout le parenchyme cérébral. D'autre part grâce à la stratégie d'isolement et de non vaccination des chiens MPS VII, nous n'observons ni de réponse immunitaire humorale, ni d'aggravation de l'inflammation du parenchyme. / Mucopolysaccharidosis type VII (MPS VII) is a rare inherited lysosomal storage disease caused by β-D-glucuronidase (β-glu) deficiency. β-glu is involved in the physiological turnover of glycosaminoglycans (GAGs). Its deficiency causes accumulation of undegraded GAGs inside vesicles leading to cell death. Our goals are to develop and test the clinical relevance of helper-dependant (HD) canine adenovirus type 2 (CAV-2) vectors to treat neural degeneration caused by MPS VII in a dog model. Because CAV-2 targets preferentially neurons and traffics via axons, the distribution of the transgene throughout the CNS will allow widespread delivery of the missing lysosomal enzyme in these disorders with a minimum number of injections. We tested a HD-CAV-2 vector expressing the human GUS gene in the canine model of MPS VII for their safety, efficacy, duration of expression and possible reversion of the MPS VII induced symptoms.A previous study based on HD-CAV-EGFP vector injections in MPS VII-/- and healthy dogs showed that we are now able to inject HD-CAV-2 in the dog brain, have a minimal induction of the immune response, an efficient transduction of the neurons and an efficient biodistribution of transduced cells. After the production of a suitable vector (HD-RIGIE) for injections in the CNS of MPS VII dogs we analysed its efficiency on GAGS storage in neurons.Injections of HD-RIGIE showed after 1 month or 4 months post injections a widespread increase in general level of β-glu activity, in the sites of injections and in distant areas such as cortex. Analysis of GFP, also permit to observe a widespread biodistribution of the vector. Because of β-glu property of cross-correction we observed a global decreased in GAGs storage in the entire MPS VII brains. Finally, the dogs did not present humoral immune response and no aggravation of inflammation

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Author: Bc. Veronika Novotná Supervisor: doc. MUDr. Josef Herink, DrSc. Title of diploma thesis: Mitochondrial dysfunction and neurodegenerative diseases The diploma thesis deals with mitochondrial dysfunction and neurodegenerative diseases and it is divided into two main parts. The first part summarized the classification of neurodegenerative diseases and general charakteristic of mitochondria.Then a describe of the processes of oxidative stress, excitotoxicity, apoptosis and briefly decribe the nervous system. The second part deals with description of mitochondrial dysfunction in selected nerodegenerative diseases. The recent studies refer to connection between mitochondrial dysfunctions and formation of neurodegenerative diseases. Keywords: excitotoxicity, mitochondrial dysfunction, neurodegenerative disorders, neuronal cell death, oxidative damage

Les encéphalopathies spongiformes transmissibles sont des maladies neurodégénératives caractérisées par une vacuolisation intense et une perte neuronale associées à l’accumulation d’une protéine prion pathologique : la PrPSc. A cause de la longue période d’incubation silencieuse de ces maladies, les individus souffrant d’une maladie à prions peuvent exposer des patients qui recevraient leur sang ou un de leurs organes à un risque de contamination iatrogène. De plus, lorsque le diagnostic survient, les dommages cérébraux sont souvent massifs, et l’issue toujours fatale. A ce jour, aucun traitement n’est disponible, et l’ensemble des stratégies testées a échoué.L’alternative développée par le laboratoire est celle de la thérapie cellulaire couplée à la thérapie génique, en utilisant des cellules souches embryonnaires (ES) délivrant des molécules anti-prions. Concernant le choix des molécules anti-prions, nous avons choisi les mutants PrP-DN. Notre hypothèse repose sur des études montrant qu’une lysine au codon 219 de la PrP chez l’Homme ou une arginine au codon 171 de la PrP ovine, protègent du développement d’une ESST. L’étude de ces mutants, en cellules infectées ou dans des souris transgéniques, a permis de montrer que les PrP mutées n’étaient pas converties en PrPSc et qu’elles exerçaient un effet protecteur dit « dominant négatif » sur la conversion de la PrPC sauvage en PrPSc.Un des projets du laboratoire avait donc pour objectif d’utiliser les cellules souches exprimant les mutants PrP-DN pour développer une stratégie de thérapie génique et cellulaire des maladies à prions : l’hypothèse étant que les cellules greffées pourraient non seulement réparer le tissu lésé mais que ce dernier serait également protégé de l’infection par les prions.Une première approche de thérapie génique et cellulaire avait été initiée au laboratoire et montrait des résultats plutôt encourageants. En effet, la greffe de cellules souches neurales murines exprimant des PrP-DN et produites à partir de cellules souches embryonnaires murines, conduisait, pour une partie des souris, à un allongement du temps d’incubation de la maladie, ainsi qu’à une diminution de l’astrogliose et de la vacuolisation.Dans ce contexte, le premier objectif de ma thèse a été de valider l’approche thérapeutique en montrant que les cellules greffées délivraient des mutants PrP-DN capables d’inhiber la réplication du prion. Nous avons opté pour un modèle de culture organotypique infectée par des prions murins. En plus de répondre aux exigences éthiques de la directive 2010/63/UE, ce modèle offre l’avantage de pouvoir réaliser plus d’essais, des cinétiques d’accumulation de PrPSc et de visualiser le devenir des cellules greffées. Enfin, opter pour cette stratégie permettait de transposer dans un modèle humanisé des travaux précédemment réalisés et ayant montré des résultats encourageants. Pour cela, il a été nécessaire de mettre en place un modèle prion ex vivo de culture organotypique de tranches de cerveau, dans lequel il était possible de réaliser des greffes et permettant d’évaluer l’effet inhibiteur des mutant-PrP-DN sur la réplication du prion. Par ailleurs, notre groupe fait partie, avec d’autres groupes travaillant avec des cellules souches mésenchymateuses, de l’équipe « Biologie des cellules souches et médecine régénératrice », il nous est apparu pertinent d’évaluer l’effet des MSC sur la pathologie prions dans des modèles prions en culture organotypique et en particulier d’évaluer l’impact de greffes de MSC concomitantes aux greffes de NSC-PrP-DN. En effet, ces cellules sont décrites comme pouvant induire un microenvironnement neuroprotecteur en limitant la prolifération des cellules de la microglie et des astrocytes, et peuvent favoriser la différenciation des NSC. Enfin notre dernier objectif visait à transposer les outils murins vers des outils « humains » en produisant des NSC humaines issues d’ES et exprimant une PrP humaine portant les mutations DN. / Transmissible spongiform encephalopathies are neurodegenerative diseases characterized by a strong vacuolization, a neuronal lost and deposits of prion pathologic protein: PrPSc. This PrPSc accumulation is the result of the conformational conversion of the host encoded endogenous PrPC protein. Although the incidence of these diseases in humans remains low (about one to two cases per million inhabitants per year), these diseases remain a public health problem. Indeed, because of their long and silent incubation period, patients with prion disease may expose people through blood transfusion or organ transplantation with a risk of iatrogenic contamination. In addition, when the diagnosis occurs, brain damage is often massive, and the outcome is always fatal and rapidly occurs. Until now, there is no treatment that could be proposed to patients.The alternative developed by our laboratory for several years, is a strategy of cell therapy coupled with gene therapy. The general objective is to use pluripotent embryonic stem cells (ESC) and graft them as a “medicine” not only to orchestrate a functional recovery of the damaged zones and protect the grafted cells from prion propagation but also to deliver anti-prion molecules.For the anti-prion molecules, we have chosen dominant negative PrP mutants (PrP-DN). Our choice is based on studies showing that a lysine at codon 219 of the human PrP or an arginine at codon 171 of the ovine PrP protect against the development of a TSE. Study of these mutants in infected cells or in transgenic mice showed that the mutated PrPC were not converted into PrPSc. Moreover, they exibit a so-called "dominant negative" protective effect on the conformational conversion of their wild-type PrPC counterparts. A first approach of gene and cell therapy was initiated in the laboratory and has shown encouraging results. Indeed, the graft of murine neural stem cells (NSC) derived from murine embryonic stem cells and expressing anti-prion molecules, has allowed, for some of the mice, to an increase the incubation time of the diseaseas well as to a decrease of astrogliosis and vacuolization.In this context, the first objective of my thesis was to validate the therapeutic approach by showing that the grafted cells were able to inhibit prion replication trough the dominant negative effect of the PrP-DN. To address this point, we have chosen to use an organotypic culture model infected with murine prions (22L strain). In addition to fill the ethical requirements under the European Directive 2010/63 and the 3Rs, organotypic culture models offer the advantage to perform and repeat experiments, kinetic tests, and PrPres analysis. This model also allows to visualize the fate of the grafted cells. Finally, by choosing this strategy, it will be possible to transpose into a humanized model the work previously performed on mouse organotypic cultures.To achieve this task, it was necessary to establish an ex vivo prion model of organotypic culture brain slices, in which it was possible to perform grafts and to evaluate the inhibitory effect of PrP-DN mutants on the prion replication.In addition, as our group is included in the team "Stem cell biology and regenerative medicine" (led by Pr Jorgensen) in which several stem cells are studied (liver stem cells and mesenchymal stem cells (MSC)) and because MSC have been shown to provide protective effects when grafted in the brain of mice with neurological diseases, it was therefore relevant to evaluate the effect of MSC on prion pathology in our prion models and in particular to evaluate the impact of concomitant MSC grafts on NSC-PrP-DN.In a last step, our goal was to transpose the mouse tools (NSC from ES and expressing the PrP-DN mutants) to "human" tools by producing human NSCs derived from human ESC and expressing human PrP-DN, and to characterized the resulted cells.

Une perturbation de la signalisation dopaminergique dans le striatum est à l’origine de troubles moteurs tels que la maladie de Parkinson (MP) ou de Huntington (MH). Une diminution de la signalisation par l’AR a été observée chez les patients atteints de troubles de la voie nigro-striée et dans des modèles de MH et MP. Nos données indiquent que l’AR synthétisé par RALDH1 et se liant au récepteur RARβ dans le striatum est nécessaire au maintien du système nigro-strié. Une perturbation de cette signalisation entraîne une diminution de l’activité mitochondriale qui conduit à une augmentation du stress oxydatif puis à l’entrée en apoptose de la cellule. Il en résulte des troubles moteurs de type MH et MP. Le rétablissement du niveau striatal de l’AR et la stimulation de RARβ par un agoniste spécifique permettent de prévenir ces phénotypes. Nos travaux nous permettent de proposer RARβ comme une nouvelle cible thérapeutique potentielle dans le cadre des neurodégénérescences de type MH et MP. / A disturbed dopaminergic signaling in the striatum leads to motor disorders such as Huntington’s (HD) and Parkinson’s (PD) diseases. A decrease of the retinoic acid (RA) signaling is observed in patients presenting disorders of the nogro-striatal pathway as well as in HD and PD models. Our data indicate that RALDH1-synthesized RA that binds the receptor RARβ in the striatum is essential to maintain the nigro-striatal system. A disturbance in this RA signaling leads to a decreased mitochondrial respiration, an increased oxidative stress and an increased apoptosis in the dorso-lateral striatum. This cellular alterations lead to HD-like and PD-like motor disorders A rescue of the striatal RA level or the stimulation of RARβ by a specific agonist prevent this phenotypes. Our work allow us to point at RARβ as a new potential therapeutic target for neurodegenerescences like HD and PD.