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Cholesterol is essential for many life processes, including correct development, fluidity of cell membranes, production of steroid hormones and bile acids, and is a major component of myelin. Smith-Lemli-Opitz Syndrome (SLOS) and Niemann-Pick disease type C (NPC) are devastating diseases and both involve dysregulation of cholesterol homeostasis. SLOS is caused by a defect in 7-dehydrocholesterol reductase (DHCR7), resulting in increased levels of 7-dehydrocholesterol (7DHC) and decreased levels of cholesterol. On the other hand, NPC is caused by defects in NPC1 or NPC2. These genes encode two lysosomal proteins that are responsible for the transport of cholesterol and other lipids out of lysosomes. Consequently, defects in these proteins results in accumulation of unesterified cholesterol within late endosomes/lysosomes. The severity ranges of both disorders are broad, and no or limited therapeutic options are available. This thesis aimed to establish the incidence and mechanisms behind SLOS and NPC in order to aid development of novel therapeutic interventions. Using a bioinformatics approach, <b>Chapter 2</b> showed that the estimated incidences of classical SLOS and NPC were similar to clinical reports. However, the analysis suggested that a late onset form of NPC1 could be more prevalent. <b>Chapter 3</b> investigated the behavioural phenotype of the SLOS Dhcr7^{T93M/&Delta;3-5} mouse model and it was found that some parallels could be drawn to the behaviour observed in SLOS patients. It also highlighted some defects in neuro-anatomy that could potentially explain certain cognitive defects. <b>Chapter 4</b> explored the suggestion of oxidative stress in the Dhcr7^{T93M/&Delta;3-5} mouse model. However, this study did not support a role for oxidative stress in this model. Genetic insights were generated via an RNAseq study on SLOS and NPC patient fibroblasts, which suggested that the WNT signalling pathway could play a role in the pathogenesis of SLOS. This pathway was also highlighted when the cells were treated with miglustat, the only approved therapy for NPC. However, the main pathway apparently affected by this drug in both SLOS and NPC was cell proliferation.

Niemann-Pick type C2 (NPC2) protein is a soluble protein that binds unesterified cholesterol. The protein helps transport unesterified cholesterol in tandem with the membrane protein Niemann-Pick type C1 (NPC1). Defects in either of proteins can cause Niemann-Pick type C disease (NPC), which results in the accumulation of unesterified cholesterol and lipids in the late endosome and lysosome. NPC is an autosomal recessive lysosomal storage disease affecting 0.35~2.20 per 100,000 people. Here we present the structural analysis of the human NPC2 glycoprotein, including expression, purification, functional analysis, homology modeling, and crystallographic studies. Human NPC2 was expressed from baculovirus-infected Trichoplusia ni (Tn5) insect cells. The construct contained a hexahistidine purification epitope tag, and the protein was purified using Nickel affinity column chromatography. The purified protein was used in binding studies with dehydroergosterol (DHE), showing that human NPC2 was functional. Using the structure of bovine NPC2, we made a homology model and mapped the human mutations onto the model. Some modeled proteins, such as the V30M and S67P variants, are unclear as to how they lead to disease, thus a structure of the human protein would be informative. Crystallization screens of human NPC2 were performed and led to crystals with a needle-like morphology, which diffracted to 4Å resolution. The structure of human NPC2 will be useful for understanding the mechanism of cholesterol binding and trafficking in cells, and to better understand the human metabolic disease NPC.

In dieser Arbeit beleuchten wir die subzelluläre Lokalisation von Flotillin unter Einfluss von Cholesterin. Wir zeigen, dass die zelluläre Lokalisation abhängig vom Cholesterinniveau der Zelle ist. Eine Cholesterindepletion bringt Flotillin in die Plasmamembran, sowie umgekehrt eine Überversorgung mit Cholesterin Flotillin in cholesterinhaltige, endosomale Strukturen führt. Dabei ist die Umverteilung abhängig von der Integrität des Zytoskeletts. Außerdem zeigt die vorliegende Arbeit, dass die Umverteilung von Flotillin von der Plasmamembran hin zu endosomalen, intrazellulären Kompartimenten abhängig vom Vorhandensein von zwei putativen Cholesterinbindungs-/Interaktionsdomänen ist. Aus den gewonnenen Daten ergeben sich weiterhin Hinweise, dass Cholesterin an Flotillin gebunden in das späte Endosomen transportiert wird. In weiterführenden Versuchen unserer Gruppe zeigte sich, dass die exosomale Freisetzung von Cholesterin bei ansteigenden zellulären Cholesterinkonzentrationen erhöht wird und dass die exosomale Cholesterinfreisetzung von Flotillin abhängig ist. Die Daten deuten auf eine mögliche Rolle von Flotillin und Exosomen bei der zellulären Cholesterinhomöostase hin.

Niemann-Pick tipo C (NPC) é uma doença de depósito lisossomal, ocasionada por alterações no tráfico de colesterol não esterificado, decorrente de alterações bialélicas nos genes NPC1 ou NPC2, ambos definindo uma doença autossômica recessiva, progressiva e irreversível, caracterizada por manifestações viscerais, neurológicas e psiquiátricas, não necessariamente combinadas. Com o propósito de descrever as características fenotípicas e genotípicas de pacientes com NPC, objetivou-se relatar dados demográficos, formas clínicas classificadas por idade, sinais e sintomas neurológicos e psiquiátricos, achados de ressonância magnética (RM) de encéfalo e ultrassonografia de abdome, assim como teste de filipin, mutações observadas e o tratamento com N-butyldeoxynojirimycin (Miglustat). De uma casuística de 12 pacientes atendidos entre 2000-2014, por revisão de prontuários, no Ambulatório de Neurogenética do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto, havia 7 mulheres e 5 homens, idade média de 20 anos (entre 2 e 42), sendo dez pacientes da etnia branca e dois mulatos, procedentes de 3 estados brasileiros: São Paulo, Mato Grosso do Sul e Minas Gerais. As formas clínicas identificadas foram infantil, juvenil e adulto. A idade do primeiro sintoma neurológico ocorreu entre 1 e 27 anos (media 9,5). Dentre os achados viscerais, dois pacientes encontravam-se assintomáticos, os demais apresentaram icterícia prolongada/colestase, hepatomegalia e esplenomegalia. Todos os pacientes apresentaram em diferentes momentos da evolução manifestações neuropsiquiátricas, tais como: paralisia do olhar vertical, ataxia/quedas, epilepsia, mioclonias, distonia, disartria, disfagia, fraqueza muscular, espasticidade, declínio cognitivo/demência, sintomas psicóticos, atraso escolar, distúrbio de comportamento, cataplexia gelástica e hipotonia neonatal. A idade de diagnóstico variou de 0 a 41 anos, com uma média de 14,5 anos. O tempo entre a idade do primeiro sintoma neurológico e o diagnóstico da doença variou de 0 a 14 anos, tempo médio de 5,3 anos. O teste de Filipin demonstrou seis resultados positivos e seis variantes. A RM de encéfalo apresentou três diferentes tipos de alteração: atrofia cerebral em 6 casos, atrofia cerebelar e desmielinização em 7. A ultrassonografia de Abdome resultou em três alterações: hepatomegalia em 8, esplenomegalia em 10 e hepatoesplenomegalia em 8. O resultado do teste genético molecular em 11 pacientes evidenciou alterações no gene NPC1 e uma paciente não possuía o resultado. A mutação c.3104C>T foi a mais frequente, em oito pacientes; c.3548G>A, de significado incerto, em um paciente, e, as demais mutações encontradas: c.3493G>A, c.3019C>G. O tratamento com N-butyl deoxynojirimycin (Miglustat) foi realizado por todos os pacientes, o tempo entre o diagnóstico e o início da medicação variou de 0 a 9 anos, média de 2,9 anos. Concluímos que o registro da doença NPC deve ser feito através de uma coleta de dados detalhada e contínua, pois sua heterogeneidade fenotípica e genotípica sugerem um número subestimado de casos, não só por sua raridade, mas também pelo desconhecimento da doença, já que há poucos grupos estudados e publicados. O seu reconhecimento precoce, associado ao adequado manejo clínico, podem retardar a progressão implacável da doença e aumentar a expectativa de vida dos pacientes. / Niemann-Pick type C (NPC) is a lysosomal storage disease caused by abnormal unesterified cholesterol trafficking, resulting from biallelic changes in NPC1 or NPC2 genes, both defining an autosomal recessive progressive and irreversible disease characterized by visceral, neurological and psychiatric manifestations, not necessarily combined. In order to describe the phenotypic and genotypic characteristics of patients with NPC, this work aimed to report demographic data, clinical forms classified by age, neurological and psychiatric signs and symptoms, brain magnetic resonance imaging (MRI) and abdominal ultrasound findings, Filipin test, the gene mutations, and the treatment with N-butyldeoxynojirimycin (Miglustat). A series of 12 patients were studied, treated between 2000-2014, by review of medical records of the Neurogenetics Clinic at the Hospital of Clinics, Ribeirão Preto Medical School, Brazil. There were 7 women and 5 men, mean age 20 years (from 2 to 42); 10 caucasian and 2 mulattos, coming from three Brazilian states: São Paulo, Mato Grosso do Sul and Minas Gerais. Infantile, Juvenile and adult clinical forms were identified. The age of the first neurological or psichiatric symptoms occurred between 1 and 27 years (mean 9.5). Among the visceral findings, two patients were asymptomatic, and the others had prolonged jaundice / cholestasis, hepatomegaly and splenomegaly. All patients had at different times of evolution symptoms, such as paralysis of vertical gaze, ataxia/falls, epilepsy, myoclonus, dystonia, dysarthria, dysphagia, muscle weakness, spasticity, cognitive decline/dementia, psychotic symptoms, school delay, disorders behavior, gelastic cataplexy and neonatal hypotonia. Age at diagnosis ranged from 0 to 41 years, with a mean of 14.5 years. The interval between the first signs of the disease and the onset of treatment ranged from zero to 14 years, with an average of 5.3 years. Filipin test resulted six positive and six variant form. The MRI scans showed three different types of changes: brain atrophy in 6 cases, cerebellar atrophy in 7 and demyelination in 7. Abdominal ultrasound revealed 8 patients with hepatomegaly, 10 with splenomegaly and 8 hepatosplenomegaly. The results of the molecular genetic testing on 11 patients showed changes in NPC1 gene and a patient did not have the result. Mutation c.3104C>T was more frequent in eight patients; c.3548G>A, of uncertain significance in a patient, and other mutations found: c.3493G>A and c.3019C>G. Treatment with N-butyl deoxynojirimycin (Miglustat) was carried by all patients; the time between diagnosis and beginning of the treatment ranged from 0 to 9, with an average of 2.9 years. We conclude that NPC disease registry should be done through a collection of detailed and continuous data because their phenotypic and genotypic heterogeneity suggest an underestimated number of cases, not only for its rarity but also by unawareness about the disease, and the fact that there are few published studies. The early recognition, coupled with appropriate clinical management, may slow the progression of the disease and increase life expectancy of patients.

La maladie de Niemann-Pick de type C (NPC) est une lipidose lysosomale complexe due à une mutation d’un des gènes NPC1 ou NPC2, qui codent pour ces protéines localisées dans les compartiments endo-lysosomaux (LE/LY). Leur absence altère le trafic intracellulaire et induit l’accumulation du cholestérol (Chol) dans les LE/LY. De plus, l’AnxA6 semble participer au transport vésiculaire du Chol en interagissant avec les microdomaines membranaires enrichis en Chol, ou avec le Chol lui-même. Dans ce travail, nous avons isolé des microdomaines membranaires résistant au Triton X-100 (également appelés DRMs pour detergent resistant membranes) à partir de lignée cellulaire NPC L1 ou de cellules saines. Les fibroblastes NPC contiennent plus de DRMs que les fibroblastes sains. Ceci semble être corrélé aux problèmes de transport du Chol dans les cellules NPC. Nous avons aussi montré qu’en présence de calcium, une partie de l’AnxA6 est associé aux DRMs, suggérant que l’AnxA6 participe à l’organisation de la membrane et par ce bias à l’étiologie de la maladie de NPC. Nous avons alors analysé les interactions de l’AnxA6-1 avec les microdomaines riches en Chol ainsi que l’implication de sa région flexible et de la séquence VAAEIL dans ces interactions. Leurs interactions avec des monocouches de Langmuir constituées de phosphatidylcholine, Chol et/ou d’acétate de cholestéryle. Nos résultats montrent que l’AnxA6 a la plus grande affinité pour les monocouches contenant du Chol ainsi que l’implication du groupement hydroxyle du Chol lors de ces interactions. / The Niemann-Pick type C (NPC) disease is a lysosomal lipid storage disorder caused by mutations in one of the two genes NPC1 or NPC2 encoding proteins of the late endosome/lysosome compartment (LE/LY). Defect in these proteins alters vesicular transport and leads to abnormal accumulation of cholesterol (Chol) in LE/LY. There are some lines of evidence suggesting that annexin A6 (AnxA6) participates in vesicular transport of Chol and may interact with membrane domains enriched in Chol and bind Chol. In this work we characterized the membrane microdomains resistant to Triton X-100, i.e., detergent-resistant membranes (DRMs) isolated from NPC patient-derived fibroblasts and from control cells. NPC cells contain a significantly higher amount of DRMs than the control cells that is consistent with the defect in Chol turnover in NPC cells. We also studied the mechanism of AnxA6 involvement in the NPC-induced changes in the membrane organization and showed that in the presence of calcium some AnxA6 molecules associate with the DRMs. This suggests that AnxA6 may play a role in the membrane lateral organization, contributing thus to the etiology of NPC disease. We then focused on the interaction of AnxA6-1 with Chol-rich membranes and on the involvement of its flexible region and VAAEIL sequence in these interactions. For this purpose, kinetics of the interfacial adsorption of human recombinant AnxA6 to Langmuir monolayers containing phosphatidylcholine, Chol and/or cholesteryl acetate were measured. Our data suggest that AnxA6 exhibits the highest affinity to Chol-containing monolayers and that the hydroxyl group of Chol plays a pivotal role in the AnxA6-lipid interactions in vitro.

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A doença de Niemann-Pick tipo C (NPC) é uma esfingolipidose autossômica recessiva que se caracteriza pelo acúmulo lisossômico de colesterol não-esterificado em vários tecidos, resultando em neurodegeneração progressiva, hepatoesplenomegalia e paralisia ocular vertical, entre outros sintomas. Sua manifestação ocorre geralmente entre a metade da infância e a adolescência. A morte ocorre geralmente até a terceira década de vida. Associado à sintomatologia clínica, o diagnóstico de NPC é realizado através do teste de Filipin em fibroblastos cultivados, que demonstra um intenso padrão de fluorescência perinuclear, consistente com o acúmulo de colesterol nãoesterificado. Alguns pacientes, referidos como NPC variantes, apresentam quadro clínico compatível com NPC, mas resultados inconclusivos nos testes bioquímicos, com fluorescência não característica no teste de Filipin, tornando problemático o diagnóstico. O objetivo deste trabalho foi desenvolver técnicas objetivas capazes de auxiliar na confirmação do diagnóstico de NPC, quando um fenótipo apresentar característica indefinida/variante ao teste de Filipin, tais como: quantificação do padrão de fluorescência perinuclear quanto ao número de pixels em pacientes com fenótipo clássico (NPC cl) e variante (NPC var) de NPC, comparando-os entre si e com controles saudáveis (CS); medida da quantidade de colesterol citoplasmático em fibroblastos dos dois grupos de pacientes; medida da atividade das enzimas esfingomielinase ácida (ASM), quitotriosidase (QT), beta-glicosidase ácida (GBA) e beta-galactosidase (GLB). Todos estes parâmetros foram comparados com aqueles de CS. Os resultados mostraram que a quantificação da fluorescência do colesterol no teste de Filipin nos três grupos estudados através do número de pixels da imagem, é um método prático e não subjetivo que demonstrou uma diferença significativa entre o acúmulo de colesterol intracelular em CS, NPC cl e NPC var, confirmando a eficácia do método para o esclarecimento de padrões duvidosos. A dosagem de colesterol intracelular em fibroblastos de NPC apresentou-se sete vezes maior no padrão clássico e quatro vezes maior no variante, do que aquela encontrada nos CS. Com esses dados, a dosagem intracelular de colesterol mostra-se um bom parâmetro quantitativo para auxiliar no teste de Filipin quando este apresentar padrão de fluorescência apenas moderado. A medida da atividade da ASM tanto em leucócitos quanto em SPF não apresentou diferenças estatisticamente significativas entre os grupos estudados, embora seja aparente a diminuição na atividade desta enzima no fenótipo NPC cl. Com os valores obtidos para a medida da atividade da QT em plasma e SPF, verificamos que não houve diferença significativa entre os dois grupos NPC mas sim entre estes e os CS, demostrando que tanto os indivíduos com o fenótipo clássico quanto com o variante possuem uma atividade aumentada desta enzima. A análise da atividade da GBA em leucócitos não apresentou diferenças significativas entre os três grupos, embora sua atividade em NPC cl pareça maior do em CS. Já na análise em SPF, esta diferença foi estatisticamente confirmada. A atividade da GLB em leucócitos não apresentou diferenças entre os grupos estudados, embora esta pareça maior nos grupos NPC do que em CS. / Niemann-Pick disease Type C (NPC) is an autosomal recessive sphingolipidosis which is characterized by a lysosomal accumulation of unesterified cholesterol in various tissues, resulting in progressive neurodegeneration, hepatosplenomegaly and vertical eye paralysis, among other symptoms. Its onset occurs commonly between middle childhood and adolescence. Death occurs usually until the third decade of life. Along with the clinical symptoms, the diagnosis of NPC is accomplished by Filipin test in cultured fibroblasts, showing an intense perinuclear staining pattern, which is consistent with the accumulation of unesterified cholesterol. Some patients, referred to as NPC variants, present a clinical picture of NPC, but inconclusive results in biochemical tests, with no characteristic fluorescence in Filipin test, which makes the diagnosis problematic. The objective of this study was to develop objective techniques that can assist in confirming the diagnosis of NPC, when the phenotype shows undefined/variant characteristics to Filipin test, such as: quantification of perinuclear fluorescence pattern based on pixels' luminosity pattern in patients with classical (NPC cl) and variant (NPC var) NPC phenotype, comparing them among themselves and with healthy controls (CS); the amount of cytoplasmic cholesterol in fibroblasts of both groups of patients; measurement of the activity of the enzymes acid sphingomyelinase (ASM), chitotriosidase (CT), beta-glucosidase acid (GBA), and beta-galactosidase (GLB). All of these parameters were compared with those of CS. The results showed that the fluorescence quantitation of cholesterol in the Filipin test, for the three studied groups, by counting the number of image pixels, is a practical and non-subjective method, which showed a significant difference between the accumulation of intracellular cholesterol in CS, NPC cl and NPC var, confirming its effectiveness to clarify dubious patterns. Measures of intracellular cholesterol in NPC fibroblasts showed seven times higher in classic and four times higher in variant pattern, than that found in CS. With these data, the dosage of intracellular cholesterol appears to be a good parameter to aid in the quantitative Filipin test when it presents only a moderate fluorescence pattern. The measurement of ASM activity in both leukocytes and in SPF did not show statistically significant differences between the groups, despite the remarkable decrease in the enzyme's activity in NPC cl phenotype. With the data obtained for QT's activity in plasma and SPF, we found that there was no significant difference between the two NPC groups, but a significant one between the NPC and the CS, demonstrating that both individuals with the classical phenotype and with the variant one have an increased activity of this enzyme. The analysis of GBA activity in leukocytes showed no significant differences among the three groups, although its activity in NPC cl seemed to be greater than in CS. In the SPF analysis, this difference was statistically confirmed. The activity of GLB in leukocytes did not differ between the groups, although it seems greater in the NPC group than in CS.