Interplay between sphingolipid and nutriente signaling in niemann-pick type C1 disease: new clues from a yeast cell model

Texto

(1)INTERPLAY BETWEEN SPHINGOLIPID AND NUTRIENT SIGNALING IN NIEMANN-PICK TYPE C1 DISEASE: NEW CLUES FROM A YEAST CELL MODEL. RITA PEREIRA VILAÇA. Tese de Doutoramento em Ciências Biomédicas. 2014.

(2)

(3) RITA PEREIRA VILAÇA. INTERPLAY BETWEEN SPHINGOLIPID AND NUTRIENT SIGNALING IN NIEMANN-PICK TYPE C1 DISEASE: NEW CLUES FROM A YEAST CELL MODEL. Tese de Candidatura ao grau de Doutor em Ciências Biomédicas submetida ao Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto.. Orientador – Doutor Vítor Costa Categoria – Professor Associado Afiliação – Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto Co-orientador – Doutora Maria Clara Pereira de Sá Miranda Categoria – Investigadora Principal Afiliação – Instituto de Biologia Molecular e Celular.

(4)

(5) V. Preceitos Legais De acordo com o disposto no nº 2 do artigo 8º do Decreto-lei nº 388/70, nesta dissertação foram utilizados os resultados de trabalhos publicados abaixo indicados. No cumprimento do disposto no referido Decreto-lei, a autora desta dissertação declara que interveio na conceção e execução do trabalho experimental, na interpretação e redação dos resultados publicados sob o nome Vilaça R.: Vilaça R, Silva E, Nadais A, Teixeira V, Matmabi N, Gaifem J, Hannun YA, Sá Miranda MC, Costa V. (2014) Sphingolipid signaling mediates mitochondrial dysfunctions and reduced chronological lifespan in the yeast model of Niemann-Pick type C1. Mol Microbiol. 91, 438-451.. Durante o tempo de execução deste trabalho, a autora desta dissertação colaborou ainda em trabalhos experimentais publicados abaixo indicados. Vilaça R, Mendes V, Mendes MV, Carreto L, Amorim MA, de Freitas V, Moradas-Ferreira P, Mateus N, Costa V. (2012) Quercetin protects Saccharomyces cerevisiae against oxidative stress by inducing trehalose biosynthesis and the cell wall integrity pathway. PLoS ONE 7(9): e45494. Teixeira V, Medeiros T, Vilaça R, Moradas-Ferreira P, Costa V. (2014) Reduced TORC1 signaling abolishes mitochondrial dysfunctions and shortened chronological lifespan of Isc1p-deficient cells. Microbial Cell. 1, 21-36..

(6) VI. O trabalho apresentado nesta tese foi realizado no Instituto de Biologia Molecular e Celular (IBMC), da Universidade do Porto, e teve o apoio financeiro do Fundo Europeu de Desenvolvimento Regional (FEDER) pelo Programa Operacional Fatores de Competitividade (COMPETE), da Fundação para a Ciência e Tecnologia (FCT) e do Programa Operacional Regional do Norte (ON.2 – O Novo Norte), através dos projectos PESTC/SAU/LA0002/2011-FCOMP-01-0124-FEDER-022718 e NORTE-07-0124-FEDER-000001 e da Bolsa de Doutoramento SFRH/BD/48125/2008..

(7) VII. [There’s nothing like the “eureka” moment of discovering something that no one knew before.] Stephen W. Hawking.

(8)

(9) IX. Agradecimentos A realização deste trabalho só foi possível com a ajuda de algumas pessoas. Aos meus orientadores, Prof. Doutor Vítor Costa e Doutora Clara de Sá Miranda, agradeço a ideia deste projecto. Ao primeiro, agradeço ainda todo o ensino e tempo dedicado ao longo destes sete anos de aprendizagem científica. Os alunos de graduação que fizeram parte do meu caminho académico: André, Alexandre, Joana e Nadais. Ao Elísio, um agradecimento especial por todo o tempo e dedicação a este projeto. Foi um prazer trabalhar contigo e desejo-te as maiores felicidades para o teu futuro, vais ser um ótimo investigador! Antigos membros e investigadores atuais no Grupo Redox Cell Signaling, pelo bom ambiente. Um agradecimento especial ao Vítor que, com o tempo, se tornou uma ajuda essencial na elaboração e compreensão deste trabalho. À Clara, pela revisão crítica desta tese. Aos membros antigos e atuais dos grupos CAM e MicroBioSyn no IBMC. Um agradecimento muito especial à Marta Mendes, que sempre me ajudou ao longo destes anos e confiou em mim na reta final. Ao grupo da UniLiPe, pela ajuda no estudo em fibroblastos. Ao Prof. Doutor Jorge Azevedo, agradeço o ponto de vista crítico e construtivo. Aos Binos (Tiggy, Xu e Lode), por tudo. Por estarem presentes, por alegrarem o meu dia. Pela amizade, sorrisos e lágrimas. Por serem especiais. Por fazerem parte do que sou e por me deixarem ser como sou (Eu escolho ser feliz!). À Joana e à Rute, pela amizade, por estarem presentes e pela força e carinho que sempre me deram. Ao resto dos pandegos, um bem haja por me animarem..sempre. Aos meus melhores amigos, aqueles que me acompanham desde a faculdade, e sem os quais isto seria definitivamente mais difícil. Joana, um enorme obrigado! Por me deixares caminhar do teu lado há tanto tempo, por seres tão especial para mim. Lígia, porque os anos passam e estás sempre do meu lado, mesmo quando seguimos vidas tão diferentes. Iva, pelo carinho e amizade ao longo destes anos. Sofia, obrigada por estares sempre presente embora distante. Ao Manu, por me deixar fazer parte da sua vida. Ao João. Pelo amor e amizade. Pela paciência e compreensão. Pela partilha. Por me tornares melhor todos os dias..

(10) X. Aos meus pais. Os meus pilares, a minha zona de conforto, o meu porto de abrigo. Devo-vos tudo e sem o vosso constante apoio moral, amor e carinho, não teria chegado tão longe. Dedico-vos esta tese e todas as minhas conquistas profissionais. Obrigada por acreditarem. Vocês são o melhor de mim! À minha pequena estrela*, que olha por mim..

(11) XI. Abstract The sphingolipid storage disorders are a group of rare diseases characterized by accumulation of lipid waste products inside the lysosomes. This can result from defects on lysosomal proteolytic enzymes or membrane transporters. The last group includes the Niemann-Pick type C (NPC) disease, characterized by a progressive neurodegenerative course that affects mainly infantile and juveniles. It is caused by loss-of-function point mutations in NPC1 (95 % of the reported cases) or NPC2. Both proteins seem to be involved in intracellular transport of endocytosed cholesterol through the endolysosomal system, but its function remains poorly characterized. NPC is characterized by an accumulation of LDLderived cholesterol inside lysosomes and a compromised homeostasis of other lipids, such as gangliosides and sphingolipids (e.g. sphingosine). Sphingolipids are important regulators of cellular processes, including vesicle trafficking and endocytosis, stress responses, cell cycle, apoptosis and senescence. However, the role of sphingosine or other bioactive sphingolipids in pathological alterations observed in NPC cells remains unknown. The NPC1 gene is highly conserved among species. The yeast Saccharomyces cerevisiae expresses Ncr1p, a NPC1 homologue present in the membrane of the vacuole. Yeast cells lacking Ncr1p have been used as a promising eukaryotic model system to characterize conserved molecular mechanisms underlying the pathophysiology of NPC1 disease. The present work aimed to study oxidative stress resistance, chronological lifespan and mitochondrial function in the yeast model of NPC1, identify cell signaling pathways deregulated in ncr1Δ mutants as consequence of changes in sphingolipids homeostasis, and characterize the role of those pathways on ncr1Δ phenotypes. Our results show that ncr1Δ mutant cells display a high sensitivity to oxidative stress induced by hydrogen peroxide that is associated with increased levels of oxidative stress markers, such as protein oxidation, lipid peroxidation and intracellular reactive oxygen species (ROS) levels, impairment of antioxidant defenses and mitochondrial dysfunctions. Taken together, these changes contribute to a shortened chronological lifespan in ncr1Δ cells. Notably, ncr1Δ cells. exhibit. major. changes. in. sphingolipids. metabolism,. including the. accumulation of long chain sphingoid bases (LCB), and sphingolipid signaling through activation of the Pkh1p-Sch9p pathway mediates ncr1Δ phenotypes. Our findings also implicate changes in the levels of ceramide, particularly increases in.

(12) XII. phytoceramide. species,. and. activation. of. the. ceramide-activated. protein. phosphatase Sit4p in mitochondrial dysfunction, oxidative stress sensitivity and shortened chronological lifespan displayed by ncr1Δ cells. Moreover, our data suggest that Sch9p- and Sit4p-dependent signaling mediate changes in the homeostasis of these lipids in both parental and ncr1Δ cells. In addition to sensing sphingolipid signals, Sch9p is activated by phosphorylation mediated by TORC1 (Target of Rapamycin complex 1), an important regulator of cell growth (nutrient sensing), ribosomal and protein turnover and cell proliferation. Our results show that TORC1 contributes to the mitochondrial dysfunctions displayed by ncr1Δ cells. Furthermore, these mutant cells exhibit a significant decrease of mitophagy, in contrast with an increase of the autophagic flux and activation of the vacuolar protease Pep4p. The overall results suggest that bioactive sphingolipids are important mediators of mitochondrial dysfunctions in the yeast model of NPC1 disease, through activation of conserved signaling pathways. The defects in mitochondria function contribute to an increased production of mitochondrial ROS, leading to the accumulation of oxidative damages, a high sensitivity to oxidative stress and a premature ageing phenotype. These studies provide new insights on signaling networks that may be deregulated in NPC cells and, therefore, may open novel avenues for potential interventions in this neurodegenerative disease..

(13) XIII. Resumo As doenças de armazenamento de esfingolípidos constituem um grupo de doenças raras caracterizadas por acumulação de produtos lipídicos tóxicos no interior do lisossoma. Isto pode ser devido a defeitos nas enzimas proteolíticas lisossomais ou nos transportadores membranares. Neste último grupo inclui-se a doença de Niemann-Pick tipo C (NPC), caraterizada por uma neurodegeneração progressiva que afeta principalmente crianças e jovens. É causada por mutações pontuais no gene NPC1 (95 % dos casos reportados) ou no gene NPC2. Ambas as proteínas parecem estar envolvidas no transporte intracelular de colesterol endocitado através do sistema endo-lisossomal, mas a respectiva função de cada proteína não está caraterizada. A doença de NPC é caracterizada por uma acumulação de colesterol derivado de LDL dentro do compartimento lisossomal e por uma homeostasia alterada de outros lípidos, como os gangliósidos e esfingolípidos, incluindo a esfingosina. Os esfingolípidos são reguladores importantes de processos celulares, incluindo o tráfico vesicular e endocitose, resposta ao stress, ciclo celular, apoptose e senescência. No entanto, o papel exato da esfingosina ou outros esfingolípidos bioactivos na patologia de NPC1 mantém-se desconhecido. O gene NPC1 é conservado entre espécies. A levedura Saccharomyces cerevisiae expressa a proteína Ncr1p, um homólogo da proteína NPC1, que está presente na membrana do vacúolo. Células com deficiência em Ncr1p constituem um bom modelo eucariótico para a caraterização de mecanismos moleculares conservados associados à patofisiologia da doença NPC1. O presente trabalho teve como objetivo o estudo da resistência ao stress oxidativo, envelhecimento cronológico e função mitocondrial no modelo de levedura de NPC1, identificar vias de sinalização desreguladas nos mutantes ncr1Δ em consequência de alterações na homeostasia de esfingolípidos e caracterizar o papel dessas vias nos fenótipos das células ncr1Δ. Os. resultados. obtidos. demonstram. que. as. células. mutantes. ncr1Δ. apresentam uma elevada sensibilidade ao stress oxidativo induzido por péroxido de hidrogénio associada a níveis elevados de marcadores de stress oxidativo, como oxidação de proteínas, peroxidação lipídica e níveis de espécies reativas de oxigénio intracelulares, diminuição de defesas antioxidantes e disfunções mitocondriais. Em conjunto, estas alterações contribuem para uma longevidade cronológica diminuída em células ncr1Δ. Particularmente, os nossos resultados.

(14) XIV. demonstram que as células ncr1Δ apresentam diferenças significativas no metabolismo de esfingolípidos, incluindo a acumulação de bases esfingóides de cadeia longa, e que a sinalização por esfingolípidos através da ativação da via Pkh1p-Sch9p medeia os fenótipos do mutante ncr1Δ. Os nossos estudos relacionam ainda alterações nos níveis de ceramida, particularmente o aumento de espécies de fitoceramida, e ativação da proteína fosfatase activada por ceramida (Sit4p) na disfunção mitocondrial, sensibilidade ao stress oxidativo e envelhecimento cronológico prematuro exibidos pelas células ncr1Δ. Além disso, os nossos dados sugerem que a sinalização dependente da proteína Sch9p e da proteína Sit4p está envolvida na regulação da homeostasia destes lípidos na estirpe parental e no mutante ncr1Δ. Além de ser um sensor de esfingolípidos, a proteína Sch9p é ativada por fosforilação mediada pelo complexo TORC1 (Target of Rapamycin complex 1), um importante regulador do crescimento celular (sensor de nutrientes), turnover de proteínas e ribossomas e proliferação celular. Os resultados obtidos demonstram que o complexo TORC1 contribui para as disfunções mitocondriais exibidas pelas células ncr1Δ. Além disso, este mutante apresenta uma diminuição significativa da mitofagia, em contraste com o aumento do fluxo autofágico e da ativação da protease vacuolar Pep4p. Os resultados apresentados nesta tese sugerem que os esfingolipidos bioativos são importantes nas disfunções mitocondriais do modelo de levedura da doença de NPC, através da ativação de vias de sinalização conservadas. Os defeitos na função mitocondrial contribuem para um aumento da produção de espécies reativas de oxigénio, levando à acumulação de danos oxidativos, uma elevada sensibilidade ao stress oxidativo e a um fenótipo de envelhecimento prematuro. Este trabalho fornece novos dados sobre as vias de sinalização que poderão estar desreguladas nas células NPC, e portanto, contribui para novas pistas para potenciais intervenções terapêuticas nesta doença..

(15) XV. Table of contents Preceitos Legais ......................................................................................................................................... V Agradecimentos ....................................................................................................................................... IX Abstract .................................................................................................................................................... XI Resumo ................................................................................................................................................... XIII Table of contents ....................................................................................................................................XV List of figures ........................................................................................................................................XVII List of tables .........................................................................................................................................XVIII Chapter 1 - General Introduction ...........................................................................................................................................1 Niemann-Pick disease .......................................................................................................................... 3 Epidemiology of NPC ......................................................................................................................... 3 Clinical description ........................................................................................................................... 4 Diagnosis ........................................................................................................................................... 5 Molecular genetics of NPC ................................................................................................................... 6 NPC proteins and functions ................................................................................................................. 8 Lipid profile of NPC ............................................................................................................................10 Impaired trafficking in NPC ...............................................................................................................12 Therapeutic approaches ....................................................................................................................13 Biological models of NPC ...................................................................................................................15 Sphingolipids ......................................................................................................................................17 Sphingolipid metabolism ................................................................................................................17 De novo biosynthesis ......................................................................................................................18 Sphingolipids turnover ...................................................................................................................20 Sphingolipid-mediated cell regulation ..............................................................................................20 Ceramide dependent cell signaling ................................................................................................21 Long-chain bases dependent cell signaling ...................................................................................24 Mitochondria, oxidative stress and ageing .......................................................................................27 ROS, antioxidant defenses and oxidative damages ......................................................................27 The free radical theory of ageing ..................................................................................................32 Lysosomal and mitochondrial function in ageing .........................................................................33 Cross-talk between nutrient and sphingolipid signaling in mitochondrial function and ageing ...36 Oxidative stress and mitochondrial involvement in Niemann-Pick disease ....................................38 Scope of this thesis ............................................................................................................................41 Chapter 2 - Sphingolipid signaling mediates mitochondrial dysfunctions and reduced chronological lifespan in the yeast model of Niemann-Pick type C1 ..........................................................43 Summary .............................................................................................................................................45 Introduction ........................................................................................................................................46 Experimental Procedures ...................................................................................................................49 Yeast strains and growth conditions .............................................................................................49 Oxidative stress resistance and chronological lifespan ................................................................50 Protein carbonylation, lipid peroxidation and intracellular oxidation .........................................51 Glutathione levels and enzymatic activities ..................................................................................51 Oxygen consumption and growth in glycerol ................................................................................52 Mitochondrial fragmentation and mitochondrial membrane potential .......................................52 Sphingolipid analysis by HPLC-MS/MS ...........................................................................................53 Protein extraction and western blotting analysis .........................................................................53 Filipin staining ................................................................................................................................54 Statistical analysis ..........................................................................................................................54 Results ................................................................................................................................................55 Ncr1p-deficient cells exhibit hydrogen peroxide sensitivity and shortened chronological lifespan associated with oxidative stress markers .....................................................................................55 ncr1Δ cells exhibit mitochondrial dysfunctions ............................................................................59 The Pkh1p-Sch9p pathway is involved in oxidative stress sensitivity, premature ageing and mitochondrial dysfunctions of ncr1Δ cells ....................................................................................62 SCH9 deletion attenuates changes in sphingolipid homeostasis of ncr1Δ cells ..........................65 Discussion ..........................................................................................................................................67.

(16) XVI Supplemental Figures ........................................................................................................................ 72 Chapter 3 - Role for ceramide accumulation and Sit4p activation in the mitochondrial dysfunctions and premature ageing of a Niemann-Pick type C1 yeast model ...................................... 75 Summary ............................................................................................................................................ 77 Introduction ....................................................................................................................................... 78 Experimental Procedures .................................................................................................................. 81 Yeast strains and growth conditions ............................................................................................. 81 Sphingolipid analysis by HPLC-MS/MS .......................................................................................... 82 Oxidative stress resistance and chronological lifespan ............................................................... 83 Enzymatic activities ....................................................................................................................... 83 Oxygen consumption and growth in glycerol ............................................................................... 83 Mitochondrial fragmentation and mitochondrial membrane potential ...................................... 84 Protein extraction and Western blotting analysis ........................................................................ 84 Statistical analysis ......................................................................................................................... 85 Results ............................................................................................................................................... 86 Ceramide levels are altered in Ncr1p deficient cells .................................................................... 86 Sit4p activity is increased in Ncr1p deficient cells ....................................................................... 88 SIT4 deletion suppresses the oxidative stress sensitivity and premature ageing of Ncr1pdeficient cells .................................................................................................................................. 89 SIT4 disruption alleviates mitochondrial dysfunctions of Ncr1p deficient cells ......................... 91 Levels of sphingolipids in sit4Δ and ncr1Δsit4Δ cells .................................................................. 93 Ceramide levels in sch9Δ and ncr1Δsch9Δ cells .......................................................................... 95 SIT4 deletion suppresses the increased levels of Sch9p and Sch9p-phospho-T570 in ncr1Δ cells ........................................................................................................................................................ 97 Discussion.......................................................................................................................................... 98 Supplemental Data................................................................................................................................ 103 Chapter 4 - Role of TORC1 in ncr1Δ phenotypes .................................................................................................. 107 Summary .......................................................................................................................................... 109 Introduction ..................................................................................................................................... 110 Experimental Procedures ................................................................................................................ 113 Yeast strains and growth conditions ........................................................................................... 113 Oxygen consumption and growth in glycerol ............................................................................. 114 Enzymatic activities ..................................................................................................................... 114 Western blotting ........................................................................................................................... 115 Statistical analysis ....................................................................................................................... 116 Results and Discussion ................................................................................................................... 117 TOR1 disruption alleviates mitochondrial dysfunctions of Ncr1p deficient cells ..................... 117 The increase of autophagic flux is conserved in the yeast model of NPC1 ............................... 118 Mitophagy is severely compromised in ncr1Δ cells .................................................................... 122 Chapter 5 - General Discussion and Future Perspectives ................................................................................ 125 References ..................................................................................................................................................................................... 135 Appendix ........................................................................................................................................................................................ 167 List of Abbreviations ....................................................................................................................... 169.

(17) XVII. List of figures Fig 1.1. Putative topology of NPC1 protein. ............................................................................................ 7 Fig. 1.2. The sphingolipid metabolism is highly conserved from yeast to mammals. ........................18 Fig. 1.3. Ceramide cell signaling. ...........................................................................................................22 Fig. 1.4. Production of reactive oxygen species in mitochondria and its detoxification by antioxidant defense systems. .....................................................................................................................................29 Fig. 1.5. Integrative regulation of Sch9p kinase in yeast by long chain sphingoid bases and nutrients. .................................................................................................................................................38 Fig. 2.1. Role of Ncr1p in hydrogen peroxide resistance......................................................................56 Fig. 2.2. Ncr1p deficiency decreases chronological lifespan. ...............................................................57 Fig. 2.3. Cytosolic catalase activity, mitochondrial superoxide dismutase activity and glutathione levels are decreased in ncr1Δ mutant cells. ..........................................................................................59 Fig. 2.4. Ncr1p deficiency decreases mitochondrial function and dynamics. ......................................61 Fig. 2.5. Levels of long-chain sphingoid bases. .....................................................................................62 Fig. 2.6. Ncr1p deficient cells exhibit increased levels of Sch9p and Sch9p-phospho-T570 and ncr1Δ phenotypes are suppressed by disruption of PKH1 or SCH9 but not by myriocin. .............................64 Fig. 2.7. Role of the LCB→Pkh1p→Sch9p pathway in mitochondrial dysfunction of ncr1Δ cells. ......66 Fig. S1. Accumulation of intracellular sterol in Ncr1p deficient cells. ..................................................72 Fig. S2. Ncr1p deficiency decreases chronological lifespan and oxidative stress resistance in W303a background. ............................................................................................................................................72 Fig. S3. S. cerevisiae BY4741 and ncr1Δ::KanMX4 cells were transformed with plasmids expressing CTT1 or SOD2 and grown to PDS phase. ...............................................................................................73 Fig. 3.1. Quantification of ceramide levels. ...........................................................................................86 Fig. 3.2. Levels of phytoceramide species. ............................................................................................87 Fig. 3.3. Levels of α-hydroxylated phytoceramide species. ..................................................................88 Fig. 3.4. The activity of Gln3p is increased in Ncr1p deficient cells.....................................................89 Fig. 3.5. SIT4 disruption suppresses oxidative stress sensitivity and shortened chronological lifespan of ncr1Δ mutant cells. ..............................................................................................................90 Fig. 3.6. SIT4 disruption suppresses mitochondrial dysfunctions exhibited by ncr1Δ cells. .............92 Fig. 3.7. The defect in mitochondrial dynamics exhibited by ncr1Δ cells is corrected by SIT4 deletion independently of mitochondrial membrane potential. ..........................................................................93 Fig. 3.8. The higher levels of Sch9p and Sch9p-phospho-T570 exhibited by ncr1Δ cells are suppressed by SIT4 deletion...................................................................................................................97 Fig. 4.1. TOR1 disruption suppresses mitochondrial dysfunctions exhibited by ncr1Δ cells. .........118 Fig. 4.2. The autophagic flux is increased in Ncr1p deficient cells. ...................................................120 Fig. 4.3. Activity of protease Pep4p is increased in ncr1Δ cells. ........................................................121 Fig. 4.4. Mitophagy is severely compromised in Ncr1p deficient cells. .............................................123 Fig. 5.1. Integrative regulation of mitochondrial function by LCBs and ceramide in Ncr1p-deficient cells. .......................................................................................................................................................132.

(18) XVIII. List of tables Table 2.1. S. cerevisiae strains used in this work. ................................................................................ 49 Table 3.1. S. cerevisiae strains used in this work. ................................................................................ 81 Table 3.2. Effect of SIT4 deletion on DHS-1-P/DHS and PHS-1-P/PHS ratios. ...................................... 95 Table S1. Levels of dihydroceramides…………………………………………………………………………..104 Table S2. Levels of phytoceramides…………...……………………………………………………….…….....105 Table S3. Levels of α-hydroxy-phytoceramides …………………………………………………………..…..106 Table 4.1. S. cerevisiae strains used in this work. .............................................................................. 113.

(19) Chapter 1. General Introduction.

(20)

(21) Chapter 1 |3. Niemann-Pick disease Lysosomal lipid storage diseases are a group of inherited metabolic disorders in which high levels of lipid compounds accumulate in lysosomes. Lysosomes are membrane-delimited organelles, described for the first time in 1955 by Christian De Duve (De Duve et al., 1955). Because they are ubiquitously presented in all mammalian cell types, except in mature erythrocytes, lysosomal diseases can affect any tissue or organ system. These diseases are responsible for shortened life expectancy although the clinical manifestations normally appear few years after birth. Niemann-Pick is designated as a group of lysosomal storage disorders with autosomal and recessive inheritance (Vanier and Millat, 2003). It was first described by Albert Niemann and Ludwig Pick as “lipid cell splenomegaly”. It was later renamed Niemann-Pick disease, remaining that designation until now. The Niemann-Pick can be classified into three subgroups according to different genetic causes and biochemical phenotypes. Type A and B result from acid sphingomyelinase deficiency (Ferlinz et al., 1991), which leads to the accumulation of high and toxic levels of sphingomyelin and other lipids in lysosomes (Brady et al., 1966). NiemannPick type C (NPC) has been mainly classified as a cholesterol transport defect (Pentchev et al., 1994) and can be subdivided in type C1 (NP-C1) or type C2 (NP-C2), according to the gene involved. This disease is characterized by abnormal cholesterol trafficking and by intracellular accumulations. of. low-density-lipoprotein. derived. cholesterol. and. glycosphingolipids in late endosomes and lysosomes (Mukherjee and Maxfield, 2004). Epidemiology of NPC The prevalence of lysosomal storage disorders is approximately 1 in 8000 births worldwide. Among this group of diseases, it was recently reported that Niemann-Pick type C (MIM, 257220) affects 0.85 in 100,000 births in Western Europe (based on Orphanet report series for the prevalence of rare diseases in Europe, November 2012). In Portugal the prevalence is higher, affecting 2.2 in 100,000 births (Pinto et al., 2004),.

(22) 4|Chapter 1. while the numbers reported for the Netherlands (0.35/100,000) and France (0.82/100,000) are significantly lower (Poorthuis et al., 1999; Vanier, 2010). However, these numbers are certainly underestimated, because the true prevalence of this disease is difficult to assess. The clinical spectrum of this disease was only recognized in the late 90’s and there is an insufficient awareness of the clinical body to this disease. The absence of a simple and rapid biochemical detection method often compromise the correct diagnosis in early ages. Clinical description The NPC disease has a very heterogeneous clinical presentation with a wide range of symptoms that are not specific for the disease. It is a progressive and fatal neurodegenerative disease with an age of onset fluctuating from perinatal period to adult age (Patterson et al., 2001). The lifespan of patients can vary between few days after birth until over 60 years old. The disease is divided in four sub-groups according with the age of onset: infantile, late infantile, juvenile and adult forms. The main feature accepted for this disease is that the age of the systemic symptoms is not associated with the neurological ones, which can appear many years after the first visceral symptoms. However, the neurological symptoms seem to be correlated with the typical course of the disease and lifespan (Vanier, 2010).. Indeed, when neurologic symptoms appear early in the life of a. patient (infantile form), the average survival is below 5 years. The neurological symptoms can include cerebellar ataxia, dysphagia, cataplexy, seizures, vertical gaze palsy, progressive dementia and death (Patterson et al., 2001). Like others neurodegenerative disorders, NPC disease is characterized by a progressive loss of neurons, particularly Purkinje cells in the cerebellum (Higashi et al., 1993) and the presence of neurofibrillary tangles (Love et al., 1995; Suzuki et al., 1995). This disease is also classified as a severe neurovisceral condition with involvement of liver, spleen and sometimes lung. Patients present an enlargement of the liver and spleen due to the presence of lipid-laden macrophages. The cells of these organs exhibit a marked cytoplasmic.

(23) Chapter 1 |5. vacuolization due to accumulation of cholesterol, phospholipids and glycolipids. Approximately 50 % of the children with NPC suffer from hepatosplenomegaly and cholestasis (Rutledge, 1989; Kelly et al., 1993). The main reason why the liver is more affected than any other tissue is that approximately 80 % of LDL-derived cholesterol and all the cholesterol from chylomicrons are removed from the circulation by the liver (Xie et al., 1999). When NPC1 is deficient, the late endosome/lysosome (LE/L) system of liver is disrupted with a massive load of exogenously-supplied cholesterol. Diagnosis Because NPC is a very heterogeneous disease, the correct diagnosis is usually very difficult to achieve. The validation of the diagnosis made by neurological evaluation always requires biochemical and molecular-genetic laboratory testing. The abnormal accumulation of cholesterol in patients with Niemann-Pick type C is used as a specific marker to diagnose this disease by filipin staining of fibroblasts. Filipin is a polyene that binds specifically to a hydroxyl group in free cholesterol to form a fluorescent complex (Schroeder et al., 1971). The accumulation of cholesterol in lysosomes is directly proportional with the filipin intensity in fibroblasts from NPC1 patients. When the filipin test is clearly positive, a specific mutation analysis is performed. This type of analysis is more accurate, but the interpretation of the results is not always simple. Due to the highly polymorphic nature of the NPC1 gene and also its length (25 exons), the identification of the disease-causing mutations is complicated. Still, some rapid methods have been described to test for the most frequent mutation I1061T (Millat et al., 1999; Millat et al., 2001b). The recommended guidelines for diagnosis of NPC were recently approved by several experts of this area (Wraith et al., 2009). Many groups are trying to develop a more sensitive and rapid test for the diagnosis of NPC disease. It was recently proposed a possible test based on the distinction of oxidized forms of cholesterol in blood samples (Porter et al., 2010), previously reported to occur in a mouse model of the.

(24) 6|Chapter 1. disease (Tint et al., 1998; Zhang et al., 2008). Also, it was reported a new method for measurement of 3β-sulfooxy-7β-N-acetylglucosaminyl-5-cholen24-oic acid and its glycine and taurine amides in urine (Maekawa et al., 2013), which was previously described to be present in the urine of NPC patients (Alvelius et al., 2001). Molecular genetics of NPC NPC can be caused by loss-of-function point mutations in either NPC1 gene (Carstea et al., 1997) or NPC2 gene (Naureckiene et al., 2000). Most of the families (about 95 % of the cases) belong to the NP-C1 group while cases of the NP-C2 group have been rarely documented. The NPC1 gene, which is localized in the chromosome 18q11-q12, encodes. for. a. large. polytopic. protein. containing. 13. putative. transmembrane domains (Fig. 1.1) that localizes mainly in the late endosomes (Ko et al., 2001; Zhang et al., 2001a) and transiently in lysosomes and the trans-Golgi network (Higgins et al., 1999; Neufeld et al., 1999). This protein is highly glycosylated in the luminal loops and presents a region (domains 3 to 7) similar to the sterol sensing domain (SSD) of other integral membrane proteins involved in cholesterol homeostasis. or. cholesterol-linked. signaling. such. as. 3-hydroxy-3-. methylglutaryl-CoA reductase (HMG-CoA), SCAP, Patched and NPC1L1 (Carstea et al., 1997; Davies and Ioannou, 2000; Park et al., 2003). This domain seems to be important for the cholesterol binding properties of NPC1 (Ohgami et al., 2004) and the regulation of cellular cholesterol trafficking (Millard et al., 2005). NPC1 also presents a cysteine-rich loop with a ring-finger motif that may be involved in protein-protein interactions (Greer et al., 1999; Watari et al., 2000; Millat et al., 2001b). The luminal Nterminal loop contains a leucine zipper motif and is called NPC1 domain, which is highly conserved among NPC1 orthologues. Recent evidences have shown that this domain is important for cholesterol binding (Infante et al., 2008b; Kwon et al., 2009; Deffieu and Pfeffer, 2011)..

(25) Chapter 1 |7. Fig 1.1. Putative topology of NPC1 protein. The Niemann-Pick type C protein contains 13 transmembrane domains (TM, grey cylinders) and 3 large hydrophilic loops facing the lumen of late endosomes and lysosomes. The three most important domains are denoted: NPC1 domain (lumenal N-terminal domain), Sterol-sensing domain (TM3-7) and the lumenal cysteine-rich loop. LE/L – Late endosomes/lysosomes.. According to the NPC variation database (Runz et al., 2008) (www. http://npc.fzk.de/), 252 sequence variants are described in the literature for NPC1 gene. The majority of disease-causing mutations are missense mutations, although more than 60 polymorphisms have also been described, and are spread throughout the NPC1 gene affecting all the functional domains (Vanier, 2010). There are few phenotype-genotype studies that correlate a mutation with the severity of disease. However, more than one-third of the described mutations are found in the cysteinerich loop, including the mutation I1061T, which is very frequent in France and UK (Millat et al., 1999). The protein with this mutation is functional but selected for endoplasmic-reticulum associated degradation, being associated with a juvenile neurological onset of NPC (Gelsthorpe et al., 2008). Several studies have shown that homozygous mutations in the SSD, including missense mutations, are very deleterious, leading to the absence of a mature and functional protein and to clinically and biochemically severe phenotypes (Yamamoto et al., 2000; Millat et al., 2001b). Indeed, expression studies of SSD mutants have reinforced the importance of this domain in NPC1 function (Watari et al., 1999). The NPC1 domain is also affected by missense mutations that seem to be associated with a severe infantile neurological form of the disease (Ribeiro et al., 2001; Sun et al., 2001)..

(26) 8|Chapter 1. The NPC2 gene, located in chromosome 14q24.3 and initially designated as HE1, encodes a small soluble glycoprotein localized in lysosomes (Naureckiene et al., 2000) that binds cholesterol with high affinity (Ko et al., 2003; Storch and Xu, 2009). Mutations in this gene are associated with severe clinical phenotypes and abnormalities in cellular cholesterol processing both in juvenile or adult patients (Millat et al., 2001a; Verot et al., 2007). Only 12 sequence variants are registered in NPC variation database (Runz et al., 2008). In the recent years, more cases of NP-C2 have been described and it has become clear that mutations in this gene also contribute to heterogeneous phenotypes as observed for NP-C1 (Vanier and Millat, 2004). NPC proteins and functions How exactly NPC1 and NPC2 function and cooperate is still one of the main questions to be solved for this disease. The NPC1 protein has been identified as an important mediator in the intracellular trafficking of LDLderived cholesterol. Cells with defective NPC1 accumulate cholesterol in lysosomes and exhibit a compromised delivery of cholesterol from LE/L to other intracellular compartments and plasma membrane (Liscum and Faust, 1987; Sokol et al., 1988; Millard et al., 2000; Wojtanik and Liscum, 2003). Zhang and co-workers have shown that NPC1 protein localizes in a particular compartment of LAMP2-containing vesicles without mannose-6phosphate receptors (Zhang et al., 2001b). Under cholesterol-depleted conditions, the NPC1 protein is widely diffused with little vesicular patterning, whereas in cells loaded with LDL-derived cholesterol the NPC1 co-localizes with LAMP-positive late endosomes or lysosomes (Higgins et al., 1999; Garver et al., 2000). Although NPC1 protein is ubiquitously expressed in all tissues, its expression is modulated by sterol metabolism. When fibroblasts cells are treated with 25-hydroxycholesterol, the levels of NPC1 mRNA decrease, whereas under cholesterol-depleted conditions or in the presence of an inhibitor of cholesterol synthesis, the levels of NPC1 mRNA increase (Watari et al., 2000; Gevry et al., 2008)..

(27) Chapter 1 |9. It was also suggested a role for NPC1 in the regulation or mediation of retrograde transport of multiple lysosomal cargo rather than sterols specifically (Kobayashi et al., 1999; Neufeld et al., 1999). Besides cholesterol,. NPC1. may also. be. responsible. for the. transport. of. gangliosides to the plasma membrane and some glycolipids from the plasma membrane (Sugimoto et al., 2001; Zhang et al., 2001b). NPC1 may function as a permease, with membrane efflux pump activity. NPC1 shares some homology with the resistance-nodulationdivision (RND) family of prokaryotic permeases and some studies have shown the ability of this protein to transport fatty acids across cellular membrane of Escherichia coli (Davies et al., 2000). However, this hypothesis was further revised and it was shown that fatty acids flux is normal in NPC1-deficient mammalian cells (Passeggio and Liscum, 2005). The NPC2 protein is structurally very different from NPC1. The mature form of the protein has only 132 aminoacids and possesses a hydrophobic pocket for specific cholesterol binding with high affinity (Ko et al., 2003; Cheruku et al., 2006; Xu et al., 2008). NPC2 depends on mannose-6phosphate receptor for lysosomal targeting and has been reported to localize. in. LAMP2-positive. LE/L. and. in. the. trans-Golgi. network. (Naureckiene et al., 2000; Blom et al., 2003). It is clear that both NPC1 and NPC2 are cholesterol-binding proteins. However, the mechanism by which both proteins function to export cholesterol out of LE/L is not yet fully characterized, although it seems that these proteins cooperate sequentially because mutations in both genes induce similar phenotypes (Vanier et al., 1996). Also, NPC1 and NPC2 double mutant mice show phenotypes similar to that of NPC1 or NPC2- single mutant mice (Sleat et al., 2004). Two potential mechanisms have been postulated for the sequential action of NPC1 and NPC2. In one model, cholesterol that accumulates in the membranes of intra-luminal vesicles of LE/L binds to NPC1 and then NPC2 acquires cholesterol from NPC1 and transfers it to the limiting membrane of LE/L. In a second model, NPC2 removes cholesterol from the intraluminal vesicles of LE/L and delivers it to the N-terminal cholesterol-binding domain of NPC1 in the.

(28) 10 | C h a p t e r 1. limiting membrane of LE/L (Kwon et al., 2009). This model was further supported by the fact that NPC2 greatly accelerates the transfer of cholesterol between phospholipid vesicles and this process is enhanced by lysobisphosphatidic acid (LBPA), whose levels are increased in both NPC1 and NPC2-deficient cells (Cheruku et al., 2006). It was suggested a possible dimerization of NPC1 to form a transmembrane channel that hypothetically allows cholesterol transport through the glycocalyx of the lysosome (Infante et al., 2008a). Lipid profile of NPC The main biochemical defect described for NPC is the accumulation of unesterified. cholesterol. in. the. late. endosomal/lysosomal. system.. Cholesterol can be produced by endogenous biosynthesis or can be obtained by hydrolysis of exogenous sources of cholesterol such as LDL. These particles are endocytosed by clathrin-coated vesicles and delivered to acidic compartments for. hydrolysis by acid lipase to provide. unesterified cholesterol (Brown and Goldstein, 1986). In normal cells, the free cholesterol is transported out of endosomes to the endoplasmic reticulum and plasma membrane. However, in NPC deficient cells, the cholesterol does not exit the endocytic pathway and accumulates within lysosomes. This was firstly observed by Pentchev and co-workers who have demonstrated that LDL-derived cholesterol is normally transported through the endocytic system to the lysosome but unesterified cholesterol is trapped due to NPC1 defects (Pentchev et al., 1985). The sequestration of unesterified cholesterol in LE/L is paralleled with defects in the distribution of cholesterol to other organelles, such as endoplasmic reticulum (ER) (Neufeld et al., 1996), Golgi complex (Coxey et al., 1993), plasma membrane. (Lusa. et. al.,. 2001;. Wojtanik. and. Liscum,. 2003). and. mitochondria (Frolov et al., 2003). The compromised movement of unesterified cholesterol to the ER, where the cholesterol homeostatic machinery is present (Goldstein et al., 2006), leads to a defect in the sensing of the cholesterol levels. Thus, despite the increase in the levels of intracellular cholesterol, the rate of cholesterol synthesis and LDL.

(29) C h a p t e r 1 | 11. receptors production are induced (Liscum and Faust, 1987), leading also to the entrapment of endogenously synthesized cholesterol (Cruz and Chang, 2000; Reid et al., 2003). Other proteins related to cholesterol trafficking can also be involved in this phenotype of NPC1 cells. For instance, the removal of cholesterol from late endosomes to the apoprotein A-I is mediated by the ABC (ATP binding cassette) transporters ABCA-1 (Chen et al., 2001) and ABCG1 (Gelissen et al., 2006), and cholesterol efflux is decreased in NPC1 cells due to a decrease in ABCA-1 protein levels (Choi et al., 2003). The neurons of the NPC patients are the primary sites that show clinical manifestation of the disease, but the brain and the central nervous system (CNS) do not uptake cholesterol in the form of LDL. Indeed, the LDL particles are blocked by the blood brain barrier and all the cholesterol present in the CNS is obtained by de novo synthesis (Dietschy and Turley, 2001). Thus, the function of NPC1 in CNS has been quite difficult to understand. Total cholesterol levels in the neurons are not significantly increased by NPC1 deficiency, but the distribution of cholesterol is impaired, being mainly present in the cell bodies (Karten et al., 2002). Consistent with these findings, the transport of endogenously synthesized cholesterol from cell bodies to distal axons in NPC1 neurons is affected (Karten et al., 2003). Also, glial cells from Npc1-/- mice exhibit accumulation of cholesterol in LE/L (Karten et al., 2005). The main storage lipid components present in the brain of NPC patients appears to be glycosphingolipids, such as GM2 and GM3 gangliosides (Vanier, 1999; Zervas et al., 2001a). GM2 also accumulates in fibroblasts (Watanabe et al., 1998). However, cholesterol quantification in neuronal cells is difficult, probably due to a mask effect resulting from the extensive loss of myelin in the brain (Xie et al., 2000; German et al., 2002). The accumulation of gangliosides in the brain seems to result from altered vesicular transport or impaired catabolism (Salvioli et al., 2004), but the clinical relevance of their accumulation is still unclear. Sphingomyelin also accumulates in LE/L of NPC1-deficient cells. The localization of lysosomal acid sphingomyelinase is abnormal in NPC1 cells (Devlin et al., 2010) and the activity of this enzyme is lower, although the.

(30) 12 | C h a p t e r 1. protein levels are normal (Elleder and Smid, 1985; Devlin et al., 2010). Since sphingomyelin and cholesterol are similarly distributed in the cell, mainly in the plasma membrane, deficiencies that affect the metabolism of one lipid generally affect the homeostasis of the other lipid. Another. lipid. that. is. accumulated. in. NPC1-deficient. cells. is. sphingosine. The levels of this sphingolipid are increased in liver, spleen and brain and this storage is unique for NPC1 (Rodriguez-Lafrasse et al., 1994). In addition, sphingosine accumulates in membrane microdomains in liver and spleen of Npc1–/– mice (te Vruchte et al., 2004). Notably, exogenously added sphingosine or sphinganine are capable of inducing NPC phenotypes in normal cells. Moreover, sphingosine is the first lipid to be elevated when normal cells are exposed to the drug U18666A (class II amphiphile compound) that induces a NPC phenotype in normal cells. Sphingosine storage affects the levels of calcium in LE/L (Lloyd-Evans et al., 2008). Because this calcium pool is important in healthy cells to mediate vesicular release and LE/L fusion events (Pryor et al., 2000), the accumulation of sphingosine in these compartments may explain the dramatic trafficking defects that are associated with NPC1 deficiency (see below). Impaired trafficking in NPC There are some evidences that cholesterol accumulation in the late endosomes can be responsible for an impairment of vesicular trafficking pathways. Endosomes contain variable amounts of cholesterol and there is a continuous vesicular membrane trafficking between the compartments of endocytic and secretory pathways where cholesterol is sorted during vesicle formation (Lebrand et al., 2002; Holtta-Vuori and Ikonen, 2006). The loss of functionality of NPC1 has been correlated with a severe impairment. of. vesicular. trafficking,. leading. to. the. formation. of. multilamelar vesicles. The normal rapid tubulovesicular movement of the late endosomes is retarded (Ko et al., 2001; Zhang et al., 2001a) and the trafficking of mannose-6-phosphate receptors is compromised (Kobayashi.

(31) C h a p t e r 1 | 13. et al., 1999). Also, the movement of LDL-derived cholesterol through the Golgi network is reduced in NPC1 fibroblasts (Coxey et al., 1993). The vesicular trafficking depends on Rab proteins, which belong to a superfamily of GTPases and are present in the membranes of the organelles that interact during the transport cycle (Feng et al., 1995). The levels of Rab9, which is involved in vesicle trafficking between late endosomes and the TGN, increase 80 % in NPC1 fibroblasts, but Rab9 is entrapped in LE/L, impairing the trafficking of mannose-6-phosphate receptors (Ganley and Pfeffer, 2006). Consistently, the overexpression of Rab9 in NPC1-deficient cells decreases cholesterol accumulation (Walter et al., 2003; Narita et al., 2005). Similar results are observed when Rab4, Rab7 and Rab8 are overexpressed in NPC fibroblasts (Choudhury et al., 2002; Choudhury et al., 2004; Linder et al., 2007). Thus, the modulation of the expression of these proteins was suggested as an approach to overcome the trafficking defects in NPC1. The Rab9 may bypass the requirement of NPC1 function, as the overexpression of Rab9 in Npc1-/mice increases lifespan and reduces ganglioside accumulation in the brain (Kaptzan et al., 2009). It was proposed that Rab9 interacts with vimentin, an intermediate filament protein present in the cytoskeleton that is phosphorylated. by. PKC.. The. accumulation. of. lipids,. particularly. sphingosine, compromises PKC activity leading to accumulation of hypophosphorylated form of vimentin (insoluble form).. This in turn. disrupts the transport of vesicles by vimentin and results in the accumulation of LDL-derived free cholesterol and other lipids in the LE/L system. Also, Rab9 is entrapped in the insoluble aggregates of vimentin contributing to a defective traffic in NPC1 cells (Walter et al., 2009). Therapeutic approaches There are no effective therapies for Niemann-Pick type C. The available therapies only ameliorate the neurological progression of the disease. Pharmacological trials have been focused mainly on the reduction of the influx of cholesterol or other complex lipids, particularly in the brain. A promisor study was performed in NPC1-deficient mice administered with.

(32) 14 | C h a p t e r 1. N-butyldeoxynojirimycin (miglustat), which is a potent inhibitor of the first step of glycolipids synthesis, including gangliosides. The survival of mice exposed to this drug increased about 20 %, with delayed appearance of neurological symptoms (Zervas et al., 2001b). Since miglustat was already approved for treatment of Gaucher disease, these findings lead to clinical trials in patients with NPC. The overall results indicated a positive clinical improvement of the neurological symptoms of adult and juvenile patients (Pineda et al., 2009; Wraith et al., 2010). In 2009, the European Union approved miglustat as a therapy for neurological symptoms in adult and pediatric patients with NPC. Another therapeutic strategy proposed was the administration of allopregnanolone. (ALLO),. because. cholesterol. is. a. precursor. of. neurosteroids in brain and the levels of pregnenolone and ALLO decrease in the brain of NPC1-deficient mice (Griffin et al., 2004). In fact, when Npc1-/- mice are injected earlier with ALLO, there is a significant delay in the onset of neurological symptoms and increased lifespan concomitant with a decrease in the levels of GM1 and GM2 gangliosides (Griffin et al., 2004). Yet, some evidences have shown that these benefits of ALLO treatment were indeed associated with the vehicle used in these studies, cyclodextrin (CD). This compound is an oligosaccharide capable of sequestering cholesterol within its hydrophobic core (Kilsdonk et al., 1995). A single injection of CD in mice can decrease the synthesis of cholesterol in liver, spleen and brain and chronic administration delayed the clinical symptoms of the disease and increased the lifespan in both NPC1 and NPC2-deficient mice (Liu et al., 2009). However, the exact mechanism of action of both ALLO and CD is still not fully understood and further studies are currently being developed to support these findings. Nevertheless, in the beginning of 2013 a clinical trial was initiated in the United States to test cyclodextrin as a possible candidate for NPC treatment. In recent years, several pieces of evidence have shown a possible connection between the up-regulation of histone deacetylases (HDAC) and NPC phenotypes. The first report has shown that transcription of NPC1 was.

(33) C h a p t e r 1 | 15. increased by a cAMP-PKA dependent mechanism associated with histone modifications (Gevry et al., 2003). Further work showed that inhibition of HDAC, which are important enzymes in the regulation of chromatin condensation. and. transcriptional. repression,. corrected. cholesterol. accumulation in neural stem cells from Npc1-/- mice (Kim et al., 2007b). Consistently, cholesterol accumulation was significantly decreased in NPC1 fibroblasts treated with HDAC inhibitors (HDACi), at least in part, due to an increase of NPC1 expression (Pipalia et al., 2011). Besides histones, HDACs can also deacetylate lysine residues on non-histone proteins like transcription factors, chaperone proteins and other signaling proteins (Konstantinopoulos et al., 2007). Consistently, treatment with HDACi induced the expression of a heat shock protein (Hsp70) in primary cortical neurons (Marinova et al., 2009). Further studies are required to fully understand. the. interplay. between. protein. acetylation. and. NPC1. phenotypes. This is a very promising therapeutic approach for NPC disease because HDACi can efficiently cross the blood-brain barrier and bypasses the main problem of the most common cholesterol quelating agents. In line with previous observations, a more recent possible therapeutic strategy for NPC is the use of molecular chaperones. This strategy is based on the fact that some disease causing-mutations affect the localization of the protein but not its function. Indeed, the protein with the most frequent mutation, I1061T, is marked for degradation in the ER, but is functional if it reaches the LE/L with the help of chemical chaperones (Gelsthorpe et al., 2008). This finding encourages further development of patient-specific chemical chaperone therapy for NPC disease. Further work is needed to identify the specific mutations that would be suitable for this treatment and also to design more specific chemical chaperones. It is clear that the complexity of NPC disease will require a combination of various treatments to have the greatest therapeutic effect. Biological models of NPC The investigation of NPC1 and NPC2 function has been possible with the development of cell and animal models of NPC deficiency. The NPC1.

(34) 16 | C h a p t e r 1. protein is highly conserved among species and loss-of-function point mutations in orthologous genes in Saccharomyces cerevisiae (Malathi et al., 2004), C. elegans (Sym et al., 2000), Drosophila (Huang et al., 2005), mice (Pentchev et al., 1986) and cats (Brown et al., 1994) also affects lipid trafficking. A very powerful tool for biochemical studies are skin fibroblasts from NPC patients and Chinese hamster ovary cells deficient in NPC1 (Yamamoto et al., 2000; Ko et al., 2001). More recently, a hypomorphic mice expressing only 0-4 % of NPC2 protein was generated and matched with NPC1-deficient mice to generate the double mutant mice (Sleat et al., 2004). The use of drugs that induce NPC phenotypes in cultured cells is also extensively used, particularly the amphiphilic amine U18666A (Lange et al., 2000). The yeast Saccharomyces cerevisiae presents an orthologue of NPC1 named NCR1 (NP-C related gene 1) that is located in chromosome 16 (Carstea et al., 1997; Malathi et al., 2004; Berger et al., 2005a). The Ncr1p shares 35 % identity with human NPC1. This homology encompasses the three most important domains found in NPC1 (NPC domain, SSD and cysteine-rich loop). Like NPC1, the Ncr1p has an ER-signal sequence, multiple. N-glycosylation. sites. and. 13-14. predicted transmembrane. domains (Malathi et al., 2004). However, Ncr1p lacks a C-terminal dileucine motif, which is consistent with differences in targeting mechanisms among organisms. To prove that both proteins share the same function, the yeast Ncr1p was modified with a proper mammalian targeting sequence (the last transmembrane domain of the human NPC1 protein) and it was shown that it corrects the cholesterol and glycosphingolipids (GSL) trafficking defects of mammalian NPC1 cells (Malathi et al., 2004). Also, of all the miscoding mutations reported in NPC1 cases, 66 % of the affected aminoacids are conserved in yeast and 50 % are identical between Ncr1p and human NPC1, including the most well described mutation I1061T (Berger et al., 2005a). Recently, yeast was used as a tool to identify possible pathways that could exacerbate the NPC1 phenotypes in order to characterize new possible targets for disease intervention (Munkacsi et al., 2011). These authors have identified 13 genes in 12 pathways that could act as possible.

(35) C h a p t e r 1 | 17. modulators of NPC1 disease. Particularly, they have shown an upregulation of HDAC genes, which is consistent with previous data (see above), and confirmed the correction of NPC1 phenotypes by treating NPC1 fibroblasts with an HDACi. The existence of NPC1 orthologues in several organisms, such as yeast, bacteria and protozoa, highlights the importance of this protein in conserved cellular processes. Some authors have raised the hypothesis that NPC1 protein may function as key regulator of the trafficking of lipophilic substrates, such as sphingolipids, rather than being a cholesterol sensor/transporter (Malathi et al., 2004). Sphingolipids Sphingolipids are a class of biologically active lipids ubiquitously expressed in cellular membranes. Besides their structural function, they are involved in the regulation of biological processes, such as cell cycle, stress responses, apoptosis, angiogenesis, differentiation and senescence. Because of this, sphingolipids have been implicated in relevant human diseases, including sphingolipidoses (Ozkara, 2004), diabetes (Fox and Kester, 2010), cancer (Ponnusamy et al., 2010), Alzheimer’s disease (van Echten-Deckert and Walter, 2012) and other neurological syndromes (Haughey, 2010). Sphingolipid metabolism The sphingolipid metabolism is highly conserved from yeast to mammalian cells (Fig. 1.2) and has been extensively studied using this simple model organism. In both mammalian and yeast cells, the sphingolipid synthesis occurs at the ER and Golgi complex, and yeast cells have homologues or orthologues of mammalian genes involved in this process. Although the metabolic pathway is conserved, the complex lipids produced are quite different chemically and structurally. However, ceramide is the central molecule of the sphingolipid metabolism in both yeast and mammalian cells..

(36) 18 | C h a p t e r 1. Fig. 1.2. The sphingolipid metabolism is highly conserved from yeast to mammals. Adapted from (Rego et al., 2014).. De novo biosynthesis The first step of de novo sphingolipid synthesis is the condensation between. serine. and. palmitoyl-CoA. in. the. ER. to. yield. 3-. ketodihydrosphingosine, catalyzed by serine palmitoyltransferase (SPT) (Nagiec et al., 1994). The 3-ketodihydrosphingosine is converted to dihydrosphingosine (DHS) by the 3-keto reductase in a NADH-dependent reaction, also in the ER (Beeler et al., 1998). The next steps diverge slightly between mammalian and yeast cells. In yeast, DHS is converted to phytosphingosine (PHS) by hydroxylation at C4 catalyzed by the Sur2p hydroxylase (Grilley et al., 1998). The long chain bases (LCBs) DHS and PHS also differ in the hydrocarbon chain length, as DHS contains 16 to 20 carbons while PHS presents 18 or 20 carbons. Both DHS and PHS can be N-acylated to dihydroceramide and phytoceramide, respectively, by ceramide synthases, Lac1p and Lag1p, which use preferentially a C26 fatty acyl-CoA (Guillas et al., 2001). The Sur2p. hydroxylase. is. also. responsible. for. hydroxylation. of.

(37) C h a p t e r 1 | 19. dihydroceramides to produce phytoceramides. DHS and PHS can also be phosphorylated by two LCB kinases, Lcb4p and Lcb5p, to yield DHS-1-P and PHS-1-P, respectively (Nagiec et al., 1998). The LCB-1-phosphates are substrates for a specific lyase, Dlp1p, which is responsible for its conversion into fatty aldehydes and ethanolamine-1-phosphate (Saba et al., 1997). This process constitutes the only exit point from the sphingolipids metabolism and is an important regulator step in the overall sphingolipid levels (Cowart and Obeid, 2007). In mammalian cells, DHS is N-acylated by ceramide synthase to form dihydroceramide before its reduction to ceramide by desaturation. There are six genes encoding for ceramide synthases that display fatty acyl chain length specificity as well as differential tissue distribution [reviewed in (Levy and Futerman, 2010)]. The ceramide can be cleaved by ceramidases to form sphingosine, which in turn can be phosphorylated to sphingosine1-phosphate. Both in yeast and mammalian cells, ceramide is transported to the Golgi complex where the synthesis of complex lipids occurs.. The. transport of ceramide from ER to Golgi proceeds by vesicular and nonvesicular. ATP-independent. processes;. however,. the. homologue. of. mammalian CERT protein, which mediates the transport of ceramide for the synthesis of sphingomyelin in the Golgi, was not identified in yeast. In mammalian cells, sphingomyelin synthetases transfer phosphorylcholine from phosphatidylcholine to ceramide, generating sphingomyelin with concomitant production of diacylglycerol (DAG). Ceramide can also be phosphorylated. to. ceramide-1-phosphate. by. ceramide. kinase. or. glycosylated by glucosyl or galactosyl ceramide synthases (Hannun and Obeid, 2008). In yeast cells, ceramide is incorporated in complex sphingolipids with a head group of inositol phosphate instead of a choline phosphate. The first complex sphingolipids formed in the Golgi is inositol phosphorylceramide (IPC) that can be mannosylated to form MIPC (mannosyl IPC). The most abundant complex sphingolipid present in yeast is mannosyldiinositolphosphorylceramide (M(IP)2C), which is synthesized.

(38) 20 | C h a p t e r 1. by the addition of another inositolphosphate group to MIPC (Beeler et al., 1997; Dickson et al., 1997b; Uemura et al., 2003). Sphingolipids turnover The turnover of sphingolipids is essential for cells survival because the deficiency in enzymes involved in the catabolism of sphingolipids leads to human diseases like sphingolipidoses. In mammalian cells, the hydrolysis of complex sphingolipids generates ceramide through the action of several hydrolases, including sphingomyelinases that degrade sphingomyelin. These enzymes are located in sub-compartments of the cell, such as plasma membrane, mitochondria-associated membranes and lysosomes (Hannun and Obeid, 2008). Ceramide can be catabolized by ceramidases to. generate. sphingoid. bases,. which. can. be. phosphorylated. into. sphingosine-1-phosphate by sphingosine kinases or N-acylated to produce new ceramides. This process seems to be important for the recycling of mammalian sphingolipids (Sultan et al., 2006). In. yeast,. ceramide. can. inositolphosphospingolipids. be. obtained. catalyzed. by. by. Isc1p,. the. hydrolysis. of. a. homologue. of. mammalian neutral sphingomyelinase that presents phospholipase-C type activity (Sawai et al., 2000). The Ydc1p and Ypc1p ceramidases are responsible for degradation of dihydroceramide and phytoceramide into DHS and PHS, respectively (Mao et al., 2000a; Mao et al., 2000b). Sphingolipid-mediated cell regulation Sphingolipid. metabolites,. such. as. ceramide,. sphingosine. and. sphingosine-1-phosphate, have been extensively studied due to their role in the regulation of biological processes. These bioactive sphingolipids have distinct effects in cells and regulate different cell fates. While high levels of ceramide and sphingosine have been implicated in apoptosis and cell cycle arrest, increased levels of sphingosine-1-phosphate generally promotes cell survival and proliferation. Thus, the balance between ceramide, sphingosine and sphingosine-1-phosphate determines cell fate (Spiegel and Milstien, 2003)..

(39) C h a p t e r 1 | 21. Ceramide dependent cell signaling Ceramide levels can increase via activation of de novo biosynthesis or breakdown of complex sphingolipids, or by inhibition of ceramidases. In mammals, the deficiency in acid ceramidase causes the accumulation of ceramide, leading to a lysosomal storage disorder known as Farber’s disease (MIM 228000) (Park and Schuchman, 2006). Ceramide has an important role in age-related diseases mainly because its concentration gradually increases with age in most mammalian cells and tissues (Nikolova-Karakashian et al., 2008; Jana et al., 2009). Ceramide has been implicated in the regulation of cellular processes such as differentiation (Xu et al., 2010), cell cycle (Bourbon et al., 2000), apoptosis (Mullen and Obeid, 2012) and senescence (Saddoughi et al., 2008), through modulation of cell signaling pathways (Figure I.3). This lipid can directly interact with specific ceramide-activated protein kinases (CAPK), ceramide-activated protein phosphatases (CAPP), cathepsin and components of cell signaling pathways. The CAPP include protein phosphatase-2A (PP2A) (Dobrowsky and Hannun, 1993), protein phosphatase-1 (PP1) (Chalfant et al., 1999), and protein phosphatase-2C (PP2C) (Perry et al., 2012). CAPPs belonging to the PP2A family are heterotrimers with two regulatory subunits (A and B) and one catalytic subunit (C). The yeast homologue of this type of CAPPs consists of regulatory subunits encoded by the genes TPD3 and CDC55 and a catalytic subunit encoded by SIT4 (Nickels and Broach, 1996). These proteins are responsible for an anti-proliferative response in yeast cells treated with ceramide, inducing growth arrest in G1 phase of the cell cycle. Sit4p plays important roles in cell cycle progression (Sutton et al., 1991), cell integrity (Angeles de la Torre-Ruiz et al., 2002), nutrient responses via TORC1 (Jiang and Broach, 1999) and drug resistance (Miranda et al., 2010)..

(40) 22 | C h a p t e r 1. Fig. 1.3. Ceramide cell signaling. Examples of ceramide-dependent modulation of several targets and signaling pathways. CAPK – Ceramide-activated protein kinase; CAPP – Ceramide-activated protein phosphatase; PP1 – Protein phosphatase 1; PP2A – Protein phosphatase 2A; ROS – reactive oxygen species.. The cathepsin D was also identified as a ceramide-binding target (Heinrich et al., 2000) and its activation triggers lysosomal apoptosis. The interaction of ceramide with cathepsin D induces autolytic cleavage of the pro-enzyme, releasing the active components of cathepsin activity. This interaction mediates the subsequent activation of downstream apoptotic signaling, such as Bid truncation and mitochondrial dysfunction (Heinrich et al., 2004). The yeast homologue of cathepsin D is Pep4p, which can be recruited to the cytosol in response to acetic acid stress and ceramide levels, is important for mitochondrial degradation during apoptotic events (Pereira et al., 2010; Rego et al., 2012). Ceramide also plays a central role in mitochondrial-dependent apoptotic events. Indeed, ceramide can accumulate in mitochondrial membranes and inhibit components of the mitochondrial respiratory chain, promoting the generation of reactive oxygen species - ROS (Gudz et al.,.