Sphingolipid metabolism, Sit4p and TORC1 in the yeast model of Niemann-Pick type C1 disease

Sphingolipid metabolism, Sit4p and TORC1 in the yeast model of Niemann-Pick type C1 disease Ivo André Silva Barros Mestrado em Bioquímica Departamento de Química e Bioquímica 2014 Orientador Professor Doutor Vitor Manuel Vieira da Costa Instituto de Ciências Biomédicas Abel Salazar, UP Instituto de Biologia Molecular e Celular, UP Co-orientadora Doutora Rita Pereira Vilaça Instituto de Biologia Molecular e Celular, UP II FCUP ICBAS Sphingolipid metabolism, Sit4p and TORC1 in the yeast model of Niemann-Pick type C1 disease FCUP ICBAS Sphingolipid metabolism, Sit4p and TORC1 in the yeast model of Niemann-Pick type C1 disease III IV FCUP ICBAS Sphingolipid metabolism, Sit4p and TORC1 in the yeast model of Niemann-Pick type C1 disease Acknowledgments Queria começar por agradecer ao professor Vitor Costa por ter permitido a realização deste trabalho no laboratório por ele liderado. As suas aulas de sinalização molecular despertaram-me o interesse para a área, e a sua orientação durante a realização de todo o trabalho aqui exposto - e principalmente as pacientes correções feitas a esta tese - permitiram que eu a conseguisse concluir da melhor forma. É um excelente professor e esteve sempre disponível quando precisei. Um grande agradecimento também à Rita Vilaça pela sua co-orientação e paciência nestes últimos tempos, que me motivaram a ser mais exigente comigo mesmo no laboratório, assim como pelas diversas discussões sobre o trabalho aqui realizado e ajuda na preparação de apresentações orais. Não podia deixar de agradecer igualmente ao Vítor Teixeira, pela orientação que me deu nos primeiros tempos no laboratório e pela ajuda e ideias fornecidas para a realização do trabalho. Aos meus colegas de mestrado Filipe Marques e Telma Martins queria agradecer pela companhia e bom ambiente proporcionados durante todo o ano, principalmente nos nossos animados almoços na cantina da faculdade, assim como à Clara Pereira, que tanto me ajudou em diferentes alturas e tantas vezes respondeu às minhas perguntas pertinentes e, na maioria das vezes, repetitivas (“Clara, qual a voltagem a que corres os géis? Etc,..”). Um grande abraço também ao Nuno de Carvalho pelas piadas e ocasionais conversas sobre música e outros assuntos, que ajudaram a que os dias no laboratório fossem mais descontraídos. Aproveito assim também para agradecer aos restantes elementos do grupo MCA, Marta, Sílvia e Rute, não só pelas discussões de cariz científico nos group meetings mas também por todos os momentos mais relaxados e pelo ambiente animado que proporcionaram em diversas ocasiões. Um abraço também para todo o pessoal do grupo BSM. Os últimos agradecimentos são inevitavelmente os de cariz mais pessoal. Os meus pais e a minha irmã são e serão sempre o grande pilar que permite que tudo isto seja possível. Os meus amigos, em especial o João Guimarães, o Garcia, o Levy e o Marcos foram uma grande ajuda nos momentos de maior stress em que a descontração era o melhor remédio. E claro, a Joana porque…bem acho que nem é preciso dizer o porquê. Obrigado! FCUP ICBAS Sphingolipid metabolism, Sit4p and TORC1 in the yeast model of Niemann-Pick type C1 disease V Resumo A Niemann-Pick tipo C (NPC) é uma doença metabólica rara com um envolvimento neurológico e visceral, caracterizada por uma acumulação anormal de lípidos (colesterol e esfingolípidos) no sistema endossomal e lisossomal. Mutações pontuais nas proteínas NPC1 ou NPC2, envolvidas no transporte de lípidos na via endocitica, parecem ser fundamentais para o aparecimento desta patologia. Células de Saccharomyces cerevisiae sem a proteína Ncr1p, o homólogo em levedura da proteína NPC1 de mamíferos, apresentam uma maior sensibilidade a peróxido de hidrogénio, um tempo de vida mais curto e disfunções mitocondriais associadas com acumulação de bases esfingóides de cadeia longa. Os mutantes ncr1Δ também acumulam diversas espécies de ceramida, levando à ativação da proteína serina/treonina fosfatase Sit4p, homologa da proteína fosfatase 6 humana. Consistentemente, verificou-se que a atividade desta fosfatase aumenta no modelo de levedura de NPC1 e a sua deleção suprime os defeitos mitocondriais e tempo de vida reduzido de células ncr1Δ. Este trabalho teve como objetivo desvendar o papel do Sit4p na regulação do metabolismo de esfingolípidos e nas disfunções mitocondriais de células ncr1Δ. Para isso avaliámos alterações na transcrição de genes que codificam Lac1p e Lag1p, componentes do complexo ceramida sintetase, e Ydc1p e Ypc1p, duas ceramidases, em células transformadas com repórteres lacZ. Os nossos resultados mostram que a expressão destes genes está aumentada em células ncr1Δ por mecanismos dependentes da Sit4p. No entanto, a deleção do LAG1 ou YDC1 nestas células não restaurou a função mitocondrial, visto que os duplos mutantes são incapazes de crescer em fontes de carbono não fermentativas. O tratamento de células ncr1Δ com miriocina, um inibidor da serina-palmitoiltransferase, também não suprimiu as disfunções mitocondriais. A autofagia é crucial para a homeostasia celular e função mitocondrial, mas está alterada em células de NPC1. Estas células também apresentam valores aumentados de catepsina D, uma protease lisossomal. Estes fenótipos estão conservados no modelo de levedura, já que células deficientes em Ncr1p apresentam um aumento basal da autofagia e da atividade da Pep4p, um homólogo da catepsina D, dependentes da Sit4p. Estes resultados sugerem que a ativação do Sit4p medeia alterações no metabolismo de esfingolípidos, autofagia e longevidade em células ncr1Δ. A Sit4p também funciona abaixo do TORC1 (Target of rapamycin complex 1), VI FCUP ICBAS Sphingolipid metabolism, Sit4p and TORC1 in the yeast model of Niemann-Pick type C1 disease um regulador do metabolismo de esfingolipidos, da autofagia e do envelhecimento em levedura. A deleção do gene TOR1 aumentou a longevidade e a resistência a peróxido de hidrogénio de células ncr1Δ. Estes resultados implicam a Sit4p e o TORC1 nas disfunções mitocondriais e envelhecimento prematuro do modelo de levedura da doença NPC1. Palavras chave: Niemann-Pick tipo C1, Ncr1p, esfingolípidos, autofagia, TORC1, Sit4p, função mitocondrial FCUP ICBAS Sphingolipid metabolism, Sit4p and TORC1 in the yeast model of Niemann-Pick type C1 disease VII Abstract The Niemann-Pick Type C (NPC) is a rare metabolic disease with neurological and visceral involvement, characterized by an abnormal accumulation of lipids (cholesterol and sphingolipids) in the late endosomal/lysosomal network. Point mutations in either NPC1 or NPC2, which are involved in lipid transport through the endocytic pathway, seem to be instrumental for the onset of this pathology. Saccharomyces cerevisiae cells lacking Ncr1p, the yeast homolog of mammalian NPC1, present higher sensitivity to hydrogen peroxide, shortened chronological lifespan and mitochondrial dysfunctions associated with the accumulation of long chain sphingoid bases. The ncr1 mutants also accumulate several ceramide species, leading to activation of the serine threonine protein phosphatase Sit4, an homolog of human protein phosphatase 6. Consistently, the activity of this phosphatase was also shown to be increased in the yeast model of NPC1, and its deletion suppressed the mitochondrial defects and shortened lifespan of ncr1 cells. This work aimed to disclose the role of Sit4p is the regulation of sphingolipid metabolism and mitochondrial dysfunctions of ncr1 cells. For that we assessed changes in the transcription of genes encoding Lac1p and Lag1p, components of the ceramide synthase complex, and of Ydc1 and Ypc1, two paralogue ceramidases, in cells transformed with lacZ reporters. Our results show that expression of these genes was increased in ncr1 cells by mechanisms dependent on Sit4p. However, the deletion of LAG1 or YPC1 in ncr1 cells did not restore mitochondrial function as double mutants were unable to grow on a non-fermentable carbon source. Treatment of ncr1 cells with myriocin, an inhibitor of serine palmitoyltransferase, also did not suppress mitochondrial dysfunctions. Autophagy is crucial for cell homeostasis and mitochondrial function but is altered in NPC1 cells. These cells also show increased levels of cathepsin D, a lysosomal protease. This is conserved in the yeast model, since Ncr1p deficient cells exhibited Sit4-dependent increases in autophagy and in the activity of Pep4p, a homolog of cathepsin D. These results suggest that Sit4p activation mediates changes in sphingolipid metabolism, autophagy and lifespan in ncr1 cells. Sit4p also functions downstream of TORC1 (Target of Rapamycin Complex 1), a regulator of sphingolipid metabolism, autophagy and aging in yeast. The deletion of TOR1 increased the chronological lifespan and hydrogen peroxide resistance of ncr1 cells. The overall results implicate Sit4p and TORC1 in the mitochondrial dysfunctions and premature aging of the yeast model of NPC1. FCUP ICBAS Sphingolipid metabolism, Sit4p and TORC1 in the yeast model of Niemann-Pick type C1 disease 1 CHAPTER 1 INTRODUCTION 2 FCUP ICBAS Sphingolipid metabolism, Sit4p and TORC1 in the yeast model of Niemann-Pick type C1 disease 1.1 Lysosomal storage diseases The late-endosome/lysosomal system comprehends a group of organelles and pathways required by cells in order to process and recycle various exogenous metabolites and macromolecules, which can then be transported to their respective cellular compartments where they will have a specific function (Winchester, Vellodi et al. 2000). An impairment of this pathway, caused by a genetic defect in a luminal or membrane protein for instance, can many times result in the accumulation of molecules in lysosomes and other vesicles. These can be lipids or proteins and their excessive storage can be highly detrimental to the cell, leading to deficiencies in any human organ or tissue. Diseases associated with these phenotypes can lead to a class of rare, inherited heterogeneous group of disorders called lysosomal storage diseases (LSDs) (Winchester, Vellodi et al. 2000). This group of diseases, which affects mostly children of a young age, has a significant incidence on the Northern Portuguese population (25 per 100 000 live births) when compared to other countries, so the study of the molecular and genetic mechanisms underlying them are of high importance (Pinto, Caseiro et al. 2004). Examples of this disease’s include Farber’s disease, Hunter syndrome and, under focus on this thesis, Niemann-Pick type C. 1.2 Niemann-Pick type C The Niemann-Pick disease, a condition first described in the late nineteen twenties by Albert Niemann and Ludwig Pick, comprehends a group of autosomal recessive disorders caused by lipid storage abnormalities in lysosomes (Vanier 2010). Although initially defined as a single pathology, it quickly became evident that its clinic expression was highly variable in patients, particularly in what regarded age of onset, lifespan and affected organs – while some presented mostly neurological symptoms, others had a more visceral (spleen, liver and sometimes the lung) involvement. These observations led Allan C. Crocker to first divide it into four main types (Crocker 1961): type A, related to an early acute central nervous system (CNS) impairment and a massive visceral accumulation of sphingomyelin; Type B, with a chronic course and no nervous system involvement; types C and D with moderate and slower course and minor visceral involvement. In terms of biochemical and molecular criteria, it became clear there was either a primary deficiency in acid sphingomyelinase (types A and B) or a defective transport and processing of low density lipoprotein-derived cholesterol (type C) (Vanier 2010). The latter is, therefore, a lipid trafficking disorder where unesterified cholesterol and other lipids accumulate in the endocytic pathway, particularly endosomes and lysosomes. FCUP ICBAS Sphingolipid metabolism, Sit4p and TORC1 in the yeast model of Niemann-Pick type C1 disease 3 Niemann-Pick type C (NPC) quickly became a significant disease model, not only to develop therapeutic applications for it but also as a mean to better understand the metabolic impact of lipid accumulation in cells. It is an autosomal recessive, neurovisceral condition, caused by accumulation of cholesterol in the liver, spleen and brain (Pentchev, Comly et al. 1985, Pentchev, Brady et al. 1994). It has an estimated prevalence of 1:150,000 individuals and a birth prevalence that ranges from 0.66 to 0.88 per 100,000 in countries like France, UK or Germany (Vanier and Millat 2003, Vanier 2010). At the cellular level, unesterified cholesterol accumulates in the late endosomal/lysosomal system, due to a trafficking defect of this sterol to other subcellular compartments. This disease also has a wide clinical spectrum at several levels: although most of the affected individuals are young children that end up dying very young, it also affects adults, and it has a great variety of symptoms. It involves not only an accumulation of unesterified cholesterol but also sphingolipids, gangliosides (mostly GM1 and GM2) and phospholipids within the cell’s endosome/lysosomal system (Chang, Reid et al. 2005). In Portugal, the incidence of this disease appears to be much higher, with 9 cases from 1986 to 2005, when compared to other countries (Pinto, Caseiro et al. 2004, Vanier 2010). 1.2.1 Symptomatology There is a great variety of symptoms and phenotypes that usually affect patients with the disease, that include hepatic, neurological or psychiatric manifestations (Vanier and Millat 2003). Typical symptoms generally include cerebellar ataxia, dysarthria, dysphagia, and progressive dementia. Cataplexy, seizures and dystonia are other quite common features, and psychiatric disturbances are frequent in late-onset patients. However, the symptomatology of the affected subject varies according to the age of onset, which is wide. Infantile manifestations of the disease include hepatosplenomegaly, which consists in the enlargement of the liver and spleen, and respiratory failure. Young children with NPC may also show a deficient development of motor skills, many never learning to walk. Older children (6-15 years) show learning disabilities and clumsiness. In adults, the manifestations are mostly neurological and psychiatric. Psychosis is not uncommon, mostly through paranoid delusions or auditory or visual hallucinations. Patients also show depressive syndrome, behavioral problems, aggressiveness, and bipolar disorders, among others (Vanier 2010). 1.2.2. Molecular genetics and the NPC proteins The onset of NPC is thought to be caused by point mutations in two genes: NPC1, which is involved in the majority of cases (95%), and NPC2, in only 5% of the 4 FCUP ICBAS Sphingolipid metabolism, Sit4p and TORC1 in the yeast model of Niemann-Pick type C1 disease cases (Steinberg, Ward et al. 1994, Vanier, Duthel et al. 1996). The discovery of these mutations led to a subdivision between Niemann-Pick C1 and C2, according to the gene involved. The loss of function of either one of these proteins seems to be the underlying cause behind the disease and its phenotypes (Yu, Jiang et al. 2014). The NPC1 gene is localized in chromosome 18q11-12 and encodes a large integral glycoprotein found predominantly in the limiting membrane of late endosomes and lysosomes (Higgins, Davies et al. 1999, Ioannou 2005). Topological models have revealed that NPC1 has thirteen transmembrane helices and three large, glycosylated loops projecting into the lumen of the lysosome (Figure 1), which seems to be responsible for the correct recycling of cholesterol in vesicles (Yu, Jiang et al. 2014). The first of these domains is designated N-terminal domain (NTD) and appears to be the site for cholesterol binding, (Infante, Radhakrishnan et al. 2008). A cluster of five membrane spanning sequences of NPC1 share homology with the sterol-sensing domain (SSD), which is capable of binding sterols and is present in several key players of cholesterol homeostasis, namely 3-hydroxy-3-methyl-glutarylCoA reductase and sterol regulatory element binding protein cleavage-activating protein (SCAP) (Davies and Ioannou 2000). This domain was later found to be required for NPC1 to bind several sterols, with point mutations in SSD diminishing this ability (Ohgami, Ko et al. 2004). NPC1 also contains a cysteine rich loop that may be important for protein-protein interactions. The defective efflux phenotype from late endosomes and lysosomes apparently drives an upregulation in cholesterol biosynthesis, which led some investigators to propose that this could also be the case for other membrane lipids like sphingolipids and gangliosides (Yiannis a Ioannou 2005). The NPC2 gene is localized in chromosome 14q24.3. It encodes a small, soluble, secreted and recaptured lysossomal glycoprotein that binds cholesterol with high affinity in a hydrophobic pocket (Vanier and Millat 2004). Mutations in either NPC1 or NPC2 seem to lead to the same pattern of lipid accumulation (Sleat, Wiseman et al.2004), which indicates that these proteins seem to act in a cooperative fashion to take cholesterol out of the lysosomes to other cellular compartments. A hypothetical model proposes that cholesterol binds NPC2 in the lumen with its hydroxyl group exposed, and then it is transferred to the N-terminal domain of NPC1 by reversing its orientation (Kwon, Abi-Mosleh et al. 2009). NPC1 seems therefore to be important for cholesterol to “enter” the membrane. FCUP ICBAS Sphingolipid metabolism, Sit4p and TORC1 in the yeast model of Niemann-Pick type C1 disease 5 Figure 1 - Model of NPC1 structure. This protein has thirteen transmembrane domains, five of which share homology to the SSD of other sterol metabolism enzymes, three large luminal loops, and a dileucine C-terminal motif (Yu, Jiang et al. 2014). 1.2.3. Lipid involvement The accumulation of unesterified cholesterol in the late endosome/lysosomal system was one of the first cellular hallmarks described for the NPC disease, observed in cultured skin fibroblasts of NPC1 patients (Pentchev, Comly et al. 1985). Usually, after endocytosis and hydrolysis of esterified cholesterol in the lysosomes, cholesterol is delivered to other cellular compartments, like the endoplasmic reticulum (ER), Golgi apparatus, and the plasma membrane. In cells lacking NPC1, this process is apparently impaired, probably due to a disruption in the transport of free cholesterol from the lysosomes to other organelles. As consequence, although cholesterol is being accumulated, the cell is not able to sense it and therefore induces the synthesis of more cholesterol. The accumulation of cholesterol can lead to altered levels of other lipids – NPC cells also accumulate sphingomyelin, glycosphingolipids and bis- (monoacylglycerol) phosphate. The accumulation of sphingomyelin seems to be due to a decreased activity of acid sphingomyelinase (Devlin, Pipalia et al. 2010). Another important phenotype is the accumulation of sphingosine in the liver and spleen of NPC patients (te Vruchte, Lloyd-Evans et al. 2004). It has also been reported that sphingosine accumulates in the acidic compartment of drug induced models of NPC1, leading to reduced levels of sphingosine-1-phosphate and low lysosomal calcium concentrations, the latter as a consequence of sphingosine accumulation (Lloyd-Evans, Morgan et al. 2008). Sphingosine is the most simple of sphingolipids, playing important roles in cell signaling (see point 1.3 below). The pattern of lipid accumulation appears to be different in the brain and visceral organs (Vanier 2010). In liver and spleen there is no predominant compound 6 FCUP ICBAS Sphingolipid metabolism, Sit4p and TORC1 in the yeast model of Niemann-Pick type C1 disease and these organs accumulate unesterified cholesterol, glycolipids, sphingomyelin, sphingosine and sphinganine (Vanier 2010). In the brain significant alterations occur in glycosphingolipids, particularly gangliosides GM2 and GM3, glucosylceramide, lactosylceramide and GA2, all in gray matter (Vanier 1999). In fact, some studies have emphasized that brain lipid alterations occur essentially in this part of the brain, with the exception of a severe loss of galactosylceramide and other myelin lipids in the white matter of patients with infantile and late-infantile form of the disease (Vanier 1999). 1.2.4. The yeast model of Niemann-Pick type C The budding yeast Saccharomyces cerevisiae is a unicellular fungus, commonly known as baker’s yeast, which has been widely used as a eukaryotic model to characterize cellular processes conserved in evolution. Yeast is genetically well defined, is easy to manipulate and has a relatively short generation time, comparing with mammalian cells. Furthermore, the publication of its complete genome sequence in April 1996 has turned yeast into a powerful tool for gene manipulation, like gene disruption, gene marking, mutation or gene dosage effects (Mager and Winderickx 2005). This has facilitated the characterization of protein function, localization and interactions as well as the biological consequences of their loss of function. Notably, 30% of human genes involved in disease have yeast orthologues, most of them being key components in metabolic pathways (Mager and Winderickx 2005). Thus, studies using yeast can contribute to our understanding of the molecular basis of diseases, including NPC. The NCR1 (NPC1-related gene 1) gene is the yeast orthologue of human NPC1 (Berger, Hanson et al. 2005). Ncr1p shares 35% sequence identity with mammalian NPC1 (Malathi, Higaki et al. 2004) and is localized in the limiting membrane of the vacuole (Zhang, Ren et al. 2004), the equivalent of mammalian lysosome. Furthermore, this protein seems to transit through the biosynthetic vacuolar proteins sorting pathway. Importantly, Ncr1p was shown to be capable of suppressing ganglioside and cholesterol accumulation in mammalian NPC1 cells, which proves the functional equivalence of Ncr1p and NPC1. The SSD, NPC and cysteine rich domains are also conserved in yeast Ncr1p and the pleckstrin homology domain has several regions of similarity (Malathi, Higaki et al. 2004). A study revealed several pathways involved in NPC1 phenotypes. Among them, there is an up-regulation of histone deacetylase genes in cells lacking this protein (Munkacsi, Chen et al. 2011). Recently, Ncr1pdeficient cells have been used as a model of NPC1 in yeast. These cells present higher sensitivity to hydrogen peroxide and reduced chronological lifespan, associated with FCUP ICBAS Sphingolipid metabolism, Sit4p and TORC1 in the yeast model of Niemann-Pick type C1 disease 7 increased levels of oxidative stress markers, decreased anti-oxidant defenses and mitochondrial dysfunctions (Vilaça, Silva et al. 2014). Namely, they have a compromised mitochondrial network in post diauxic shift (PDS) phase, and reduced mitochondrial membrane potential, oxygen consumption and cytochrome c oxidase activity (COX). These seem to be associated with alterations in sphingolipid levels, particularly ceramides and long chain bases, a class of bioactive lipids discussed below in section 1.3. 1.3. Sphingolipids Although lipids were initially seen as being only involved in membrane structure and energy metabolism, the discovery that they could modulate cellular responses and play a role in signalling changed the way they were seen by biologists and biochemists. Sphingolipids are bioactive membrane lipids that play important signaling roles in eukaryotes, regulating cellular processes such as apoptosis, senescence, cell growth and proliferation (Dickson 2008, Hannun and Obeid 2008). Ceramide, sphingosine and sphingosine-1-phosphate (S1P) are the most studied sphingolipids, but their intricate metabolism and the several modifications they can endure gives rise to many different forms (see 1.3.1). They are also involved in certain inflammatory processes and many pathologies, like cancer (Ogretmen and Hannun 2004). Their structure is rather particular - they are composed of a long chain sphingoid base (LCB), usually a sphingosine with 18 carbons, which can be amidated with a fatty acid to generate ceramide (Figure 2). Ceramide is the precursor of complex sphingolipids such as sphingomyelin and glycosphingolipids, which are formed by addition of a polar head group (phosphocholine) or a sugar moiety, respectively (Malagarie-Cazenave, Andrieu-Abadie et al. 2004). Ceramides can also be hydroxylated and exist in saturated and non-saturated forms. Figure 2 - General structure of ceramide. Ceramide, one of the core sphingolipids in mammals and yeast, is composed of sphingosine amidated to a fatty acid of variable length. This sphingolipid is actually a family of more than 50 species (Hannun and Obeid 2008). 8 FCUP ICBAS Sphingolipid metabolism, Sit4p and TORC1 in the yeast model of Niemann-Pick type C1 disease 1.3.1. Metabolism of sphingolipids in yeast The de novo synthesis of LCBs and ceramide The de novo synthesis of sphingolipids begins in the ER with the condensation of palmitoyl-CoA with serine, in a reaction catalyzed by serine palmitoyltransferase (SPT), a membrane associated enzyme, to yield 3-keto-dihydrosphingosine (KDS) (Hanada 2003) (Figure 3). This enzyme is encoded by LCB1 and LCB2 in yeast (Lee, Lee et al. 2014, Martin, Flandez et al. 2005). Besides Lcb1p and Lcb2p, SPT also requires a third subunit, Tsc3p, for optimal activity (Gable, Slife et al. 2000). This step represents the only entry point and the first rate-limiting step in this pathway. Regulation of SPT controls the rate of sphingolipid synthesis and it can be pharmacologically inhibited by myriocin (Wadsworth, Clarke et al. 2013). Figure 3 - Sphingolipid metabolism in yeast. In Saccharomyces cerevisiae, sphingolipid metabolism is spatially divided between the Endoplasmic Reticulum (ER) and the Golgi apparatus (for complex sphingolipids). The first entry point is through SPT, and the only exit point is catalyzed by Dpl1p (Rego, Trindade et al. 2012). In S. cerevisiae KDS is then reduced to dihydrosphingosine (DHS) by Tsc10p, in a reaction that requires NADPH as a reducing agent. DHS in turn can be hydroxylated by FCUP ICBAS Sphingolipid metabolism, Sit4p and TORC1 in the yeast model of Niemann-Pick type C1 disease 9 Sur2p or Syr2p to yield phytosphingosine (PHS). Dihydrosphingosine and phytosphingosine are the long chain bases present in yeast and they can be either phosphorylated or used to generate ceramide species. Dihydrosphingosine and phytosphingosine can be amide linked to a C26 fatty acid to generate dihydroceramide (DHC) and phytoceramide (PHC), respectively. This reaction is catalyzed by the ceramide synthases Lac1p (longevity assurance gene 1 cognate) and Lag1p (longevity assurance gene 1) (Guillas, Kirchman et al. 2001, Schorling, Vallée et al. 2001). Lac1p and Lag1p form a heteromeric complex with Lip1p which is required for optimal ceramide synthase activity (Vallée and Riezman 2005). Phytoceramide can also be generated by the hydroxylation of dihydroceramide catalyzed by Sur2p (Haak, Gable et al. 1997). At this point, phytoceramide and dihydroceramide can be hydrolyzed back to LCBs and a free fatty acid in a reaction catalyzed by Ypc1p or Ydc1p, respectively (Mao 2000, Mao, Xu et al. 2000). YDC1 and YPC1 are homologous genes that encode alkaline ceramidases in yeast. Ypc1p even has reverse ceramide synthase activity (Mao 2000). The LCBs here generated can then be phosphorylated by Lcb4p or Lcb5p to yield their phosphorylated analogous, DHS-1-phosphate or PHS-1-phosphate. These species can be converted to non lipidic species (hexadecenal or phosphoethanolamine) through the only exit point in sphingolipid metabolism in yeast, catalyzed by dihydrosphingosine-1phosphate lyase (DPL1p) (Saba, Nara et al. 1997). Synthesis and hydrolysis of complex sphingolipids Ceramides can also be transported to the Golgi apparatus where they are used for the synthesis of complex sphingolipids, through the addition of different polar head groups (Dickson 2008). The inositol phosphoryl ceramide synthase, encoded by AUR1, adds myo-inositol phosphate to the C1 hydroxyl group of ceramide, to yield inositol phosphorylceramide (IPC). Then, IPC can be mannosylated by the transfer of mannose from GDP-mannose to form mannose-inositol-phosphorylceramide (MIPC), in a reaction catalyzed by the inositol phosphoceramide mannosyl transferase. This enzyme can be present in two protein complexes that contain a regulatory subunit (Csg2p) and one catalytic subunit (Csg1p or Csh1p). The last and most abundant of the complex sphingolipids in yeast, mannose-diinositol-phosphorylceramide (M(IP)2C), is generated through the addition of another inositol-phosphate moiety to MIPC, catalyzed by the enzyme inositol-phosphotransferase (Ipt1p), (Dickson, Nagiec et al. 1997). These complex sphingolipids are present in the Golgi apparatus, vacuole and 16 FCUP ICBAS Sphingolipid metabolism, Sit4p and TORC1 in the yeast model of Niemann-Pick type C1 disease 1.4.2. Regulation of mitochondrial function and longevity TORC1 is also involved in stress response, mitochondrial function and aging – treating mice with rapamycin, even if at later stages, was shown to extend the lifespan in mice. In yeast, TORC1 is active during logarithmic phase and inhibits Rim15p, a protein kinase required for the activation of the transcription factors Gis1p and Msn2p/4p, which upregulate genes associated with stress response and CLS (Wanke, Pedruzzi et al. 2005, Wei, Fabrizio et al. 2008). In fact, the deletion of TOR1 increases CLS in yeast (Powers III, Kaeberlein et al. 2006, Pan, Schroeder et al. 2011). In early stages of growth, Tor1p deficient cells have an increased mitochondrial oxygen consumption that increases the production of superoxide radicals. These ROS induce an adaptive response that allows cells to maintain ROS levels at low levels in stationary phase, increasing the CLS of tor1 mutants (Bonawitz, Chatenay-Lapointe et al. 2007). TORC1 regulates lifespan and oxidative stress resistance in part through modulation of Sch9p. Indeed, the deletion of SCH9 also improves mitochondrial function, oxidative resistance and lifespan in yeast (Pan and Shadel 2009). 1.4.3. Regulation of autophagy Autophagy is a highly regulated cellular process in eukaryotes, conserved from yeast to mammals, that plays essential roles in the regulation of normal development and cellular homeostasis. In this process, cellular proteins and whole organelles are engulfed by cytoplasmic double-membrane vesicles, designated as autophagosomes, which then direct the cargo to lysosomes where they are degraded (Klionsky and Emr 2000). In S. cerevisiae, this degradation step occurs in the vacuole, the yeast equivalent to lysosomes in mammalian cells. This process also represents an adaptive catabolic response of the cell in order to acquire nutrients and energy from more complex molecules, in response to nitrogen starvation or other types of cellular stress (Nakatogawa, Suzuki et al. 2009, Yang and Klionsky 2010). Its impairment is known to be an underlying cause behind several pathologies in humans, like cancer, immune disease and neurodegeneration, and has been implicated in aging (Levine and Kroemer 2008). It may lead to the accumulation of unfolded and toxic proteins, among other molecules. The autophagic flux seems to be defective in NPC1 cells, due to impaired degradation of autophagosomes (Sarkar, Carroll et al. 2013), and the restoration of this process may be a therapeutic approach to this problem (Sarkar, Maetzel et al. 2014). Although initially seen as a non-selective process (macroautophagy), different types of autophagy are responsible for the degradation of different organelles in a more FCUP ICBAS Sphingolipid metabolism, Sit4p and TORC1 in the yeast model of Niemann-Pick type C1 disease 17 selective fashion (Fimia, Kroemer et al. 2013, Kraft, Peter et al. 2010). In microautophagy, the lysosomal membrane directly invaginates the cargo to be degraded, independently of autophagosome formation. In mitophagy, only damaged or aged mitochondria are directed for degradation. This selective type of autophagy is of high importance for cells to maintain a good mitochondrial fitness, and represents a defense against oxidative stress and aging (Ding and Yin 2013). Pexophagy consists on the selective autophagic degradation of peroxisomes, in situations where the abundance of this organelle is not necessary anymore (Sakai, Oku et al. 2006). The cytoplasm to vacuole targeting (Cvt) pathway, specific to yeast, is responsible for delivering hydrolases like aminopeptidase 1 and α-mannosidase to the vacuole, and represents the only biosynthetic pathway that uses autophagy machinery (Lynch-Day and Klionsky 2010). The molecular mechanisms underlying autophagy have been extensively studied in yeast where thirty-five autophagy related genes (ATG) have been identified. This complex machinery is essential for the autophagic pathways present in yeast: macroautophagy, Cvt, pexophagy and mitophagy. Only 15 ATG genes encode the core machinery essential for the all subtypes of autophagy (Nakatogawa, Suzuki et al. 2009). These are divided into 5 different subgroups, each with a specific function. One of these essential genes is ATG8. This gene encodes an ubiquitin-like protein with homology to mammalian microtubule-associated protein. It serves as a molecular marker for membrane dynamics during autophagy, since it is localized on the isolation membranes of autophagosomes (Kirisako, Baba et al. 1999). It is associated to phosphatidylethanolamine and it mediates tethering and hemifusion of liposomes in response to lipidation, driving the growth of autophagosomes (Nakatogawa, Suzuki et al. 2009). TORC1 plays a key role in the regulation of autophagy in response to nutritional, hormonal and metabolic signals. In yeast, TORC1 negatively regulates macroautophagy through the hyperphosphorylation of Atg13p, which is involved in the first steps in the activation of this process (Figure 7) (Wullschleger, Loewith et al. 2006, Nakatogawa, Suzuki et al. 2009). In starvation like or stress conditions, the inhibition of TORC1 leads to the dephosphorylation of Atg13p, allowing its association with the Ser/Thr protein kinase Atg1p. This complex then interacts with other core Atg proteins, namely Atg17p, Atg29p and Atg31p, recruiting them to the pre-autophagosomal structure, necessary for the initiation of autophagy (Nakatogawa, Suzuki et al. 2009). 18 FCUP ICBAS Sphingolipid metabolism, Sit4p and TORC1 in the yeast model of Niemann-Pick type C1 disease Figure 7 - The role of TORC1 in autophagy regulation in yeast. The inactivation of TORC1 leads to the dephosphorilation of Atg13p and consequent association with other Atg proteins, leading to the pre-autophagosomal structure organization. 1.4.4. TOR signaling and sphingolipid metabolism The TOR pathway is also known to regulate sphingolipid synthesis at several levels. TORC1 inhibition stimulates the synthesis of complex sphingolipids downstream of SPT, through Npr1p-dependent phosphorylation of Orm1p and Orm2p (Shimobayashi, Oppliger et al. 2013). Rapamycin treatment leads to the activation of PP2A catalytic subunit Sit4p which in turn activates Npr1p, leading to Orm1/2p activation and synthesis of complex sphingolipids. The Orm1/2p also bind to and inhibit SPT, modulating the de novo sphingolipid biosynthesis (Breslow, Collins et al. 2010). In conditions of sphingolipid depletion (eg., upon treatment with myriocin), TORC2 activates Ypk1p which in turn phosphorylate and inhibit the Orm proteins, promoting SPT activation and sphingolipid synthesis (Roelants, Breslow et al. 2011). TORC2 also controls ceramide synthase activity (Aronova, Wedaman et al. 2008, Dickson 2008). In response to growth signals, TORC2 phosphorylates the protein kinase Ypk2p, which if also phosphorylated by Pkh1/2p, activates ceramide synthase. This process is also enhanced by LCBs since these are known activators of Pkh1/2p. The protein phosphatase calcineurin has the opposite effect by downregulating ceramide synthase activity, in response to heat and other stresses (Aronova, Wedaman et al. 2008). FCUP ICBAS Sphingolipid metabolism, Sit4p and TORC1 in the yeast model of Niemann-Pick type C1 disease 19 20 FCUP ICBAS Sphingolipid metabolism, Sit4p and TORC1 in the yeast model of Niemann-Pick type C1 disease CHAPTER 2 AIM OF THE WORK FCUP ICBAS Sphingolipid metabolism, Sit4p and TORC1 in the yeast model of Niemann-Pick type C1 disease 21 Recently, it was found that an increase in the levels LCBs in ncr1Δ cells leads to the hyperactivation of the Pkh1/2p-Sch9p pathway, resulting in the several mitochondrial dysfunctions and reduced lifespan of these mutants (Vilaça, Silva et al. 2014). TORC1 is a known activator of Sch9p through phosphorylation of its C-terminal moiety (Urban, Soulard et al. 2007), and this pathway is involved in aging in response to nutrients (Powers III, Kaeberlein et al. 2006, Pan, Schroeder et al. 2011). For this reason, we hypothesized genomic deletion of TOR1 could lead to an increase in lifespan and resistance to hydrogen peroxide in Ncr1p-deficient cells. Moreover, the ceramide activated protein phosphatase Sit4p is activated in ncr1Δ cells, which. is correlated with the accumulation of specific ceramide species (Vilaça et al. unpublished). The autophagic flux and Pep4p activity are also increased in these cells (Vilaça et al. unpublished). However, the role of ceramide, Sit4p and Tor1p in ncr1Δ phenotypes is poorly characterized. This work aimed to assess the role of Sit4p in the regulation of sphingolipid metabolism, autophagy and vacuolar function in ncr1Δ cells, as well as how Tor1p modulates oxidative stress resistance and chronological lifespan in this model of NPC1 disease. 22 FCUP ICBAS Sphingolipid metabolism, Sit4p and TORC1 in the yeast model of Niemann-Pick type C1 disease CHAPTER 3 MATERIALS AND METHODS FCUP ICBAS Sphingolipid metabolism, Sit4p and TORC1 in the yeast model of Niemann-Pick type C1 disease 23 3.1. Yeast strains and growth conditions Saccharomyces cerevisiae BY4741 was the parental strain of all haploid mutants used in this study, which are listed in table 1. Yeast cells were grown aerobically at 26ºC in a shaker at 140 rpm, in flasks with a 1:5 ratio of volume/medium, to either logarithmic (OD600 = 0.6) or post-diauxic shift (OD600 = 7-10) phase. Growth media used were yeast extract peptone dextrose (YPD - 2% (w/v) bacteriological peptone, 1% (w/v) yeast extract, 2% (w/v) glucose), yeast extract peptone glycerol [YPG - 2% (w/v) bacteriological peptone, 1% (w/v) yeast extract, 3% (w/v) glycerol), minimal medium [MM - 0.67% (w/v) yeast nitrogen base without amino acids, 2% (w/v) glucose, supplemented with 0.004% (w/v) histidine, 0.008% (w/v) leucine, 0.004% (w/v) methionine or 0.004% (w/v) uracil] and synthetic complete drop-out medium [SC - 2% (w/v) glucose, 3% (w/v) glycerol or 2% (w/v) galactose and 0.67% (w/v) yeast nitrogen base w/o amino acids, 0.14% drop out medium, supplemented with 0.008% (w/v) histidine, 0.04% (w/v) leucine, 0.008% (w/v) tryptophan and 0.008% (w/v) uracil]. 24 FCUP ICBAS Sphingolipid metabolism, Sit4p and TORC1 in the yeast model of Niemann-Pick type C1 disease Table 1. Yeast cells used in this work. Strain Genotype Source BY4741 Mata, his3Δ, leu2Δ, met15Δ, ura3Δ EUROSCARF ncr1Δ::KanMX4 BY4741 ncr1Δ::KanMX4 Vilaça et al., 2014 ncr1Δ::URA3 BY4741 ncr1Δ::URA3 Vilaça et al., 2014 sit4Δ BY4741 sit4Δ::HIS3 Vilaça et al., unpublished ncr1Δsit4Δ BY4741 ncr1Δ::KanMX4 sit4Δ::HIS3 Vilaça et al., unpublished tor1Δ BY4741 tor1Δ::KanMX4 Teixeira et al., 2014 ncr1Δtor1Δ BY4741 ncr1Δ::URA3 tor1Δ::KanMX4 This study ydc1Δ BY4741 ypc1Δ::KanMX4 EUROSCARF ypc1Δ BY4741 ydc1Δ::KanMX4 EUROSCARF lag1Δ BY4741 lag1Δ::KanMX4 EUROSCARF ncr1Δypc1Δ BY4741 ncr1Δ::URA3 lag1Δ::KanMX4 Vilaça et al., unpublished ncr1Δydc1Δ BY4741 ncr1Δ::URA3 ydc1Δ::KanMX4 This study ncr1Δlag1Δ BY4741 ncr1Δ::URA3 lag1Δ::KanMX4 This study 3.2. Genomic DNA extraction Yeast cells were grown overnight in 10 ml YPD to stationary phase, pelleted and resuspended in 100 µl of lysis solution (2% (v/v) triton X-100, 1% (w/v) SDS, 100 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA). Cell lysis was performed by adding zirconium beads, 50 µl of phenol and 50 µl of chloroform/isomyl alcohol solution (48:1) followed by a subsequent 5 min vortexing step. After centrifugation (13,000 rpm, 5 min), the aqueous phase was transferred to a new tube containing 100 µl of chloroform and FCUP ICBAS Sphingolipid metabolism, Sit4p and TORC1 in the yeast model of Niemann-Pick type C1 disease 25 100 µl of TE solution (100 mM Tris, 10 mM EDTA pH 8.0), which was then vortexed during 5 min. Afterwards, the aqueous phase was collected again to a new tube to which 1 mL of 100% ethanol was added. The mix was left at -20ºC for 5-10 min until formation of a precipitate and centrifuged at 14,000 rpm during 5 min. The pellet was resuspended in 400 µl of TE buffer and 30 µl of 1 mg/ml RNase A, and incubated at 37ºC during 7 min. Then 10 µl of 4 M ammonium acetate and 500 µl of 100% ethanol were added, and the solution mixed. After centrifugation, the pellet was washed once with 200 µl of 70% (v/v) ethanol, centrifuged again, dried and resuspended in 50 µl of dH2O. Genomic DNA was then quantified using a Nano-drop spectrophotometer (ND1000, Thermo Scientific). 3.3. Polymerase chain reaction Polymerase chain reaction (PCR) was performed with a reaction mix containing 1X Taq buffer (Promega), 0.2 mM dNTP’s (Thermo Scientific), 0.2 µM forward primer, 0.2 µM reverse primer, 1.5 mM MgCl2 (Promega) and 1 U Taq Polymerase (Promega), to a final volume of 20 µl. The primers used in this study are listed in table 2. For the amplification of the KanMX4 cassette used in the disruption of YDC1, the annealing temperature was 53ºC and the elongation time 2 min. The KanMX4 cassette also used for the disruption of LAG1 was amplified using an annealing temperature of 50ºC and an elongation time of 2 min. For the amplification of the HIS3 cassette used in the disruption of YDC1, the annealing temperature was 58ºC and the elongation time 1 minute. For the amplification of the URA3 cassette used in the disruption of NCR1, the annealing temperature was 53ºC and the elongation time 2.5 min. All PCR products were analyzed by nucleic acid electrophoresis at 100V, using 1% (w/v) agarose gels and TAE buffer 1x (40mM Tris, 20mM acetic acid, 1mM EDTA). DNA bands were extracted from the gel using the Gel Band Purification Kit (GE Healthcare, Life Sciences). 32 FCUP ICBAS Sphingolipid metabolism, Sit4p and TORC1 in the yeast model of Niemann-Pick type C1 disease 4.1. Sphingolipid metabolism in ncr1Δ cells 4.1.1. Sit4p regulates sphingolipid metabolism in the yeast model of NPC1 Aiming to characterize how sphingolipid metabolism is affected in ncr1Δ cells and the role of Sit4p in those changes, the expression of genes encoding for ceramide synthases (LAC1 and LAG1) or ceramidases (YPC1 and YDC1) was measured in BY4741, ncr1Δ, sit4Δ and ncr1Δsit4Δ cells transformed with a LacZ reporter under the control of the respective promoters. The analysis of β-galactosidase activity showed that the expression of the genes encoding for the ceramide synthases Lac1p and Lag1p increased in ncr1Δ cells when compared to parental cells (Figure 8). This increase was mostly noted for LAG1, which presented a 10-fold upregulation while LAC1 expression only increased 2-fold. Importantly, the deletion of SIT4 in ncr1Δ cells decreased the expression of these genes to levels similar to those observed in parental cells. Figure 8 - Expression of ceramide synthase genes are increased in ncr1Δ cells by a Sit4p-dependent mechanism. S. cerevisiae BY4741, ncr1Δ, sit4Δ and ncr1Δsit4Δ cells carrying LacZ reporter fusions with the promoters of LAG1 or LAC1 in the multicopy plasmid YEp357 were grown to log phase (OD600=0.6) in SC-medium lacking uracil. - Galactosidase activity was measured spectophotometrically, as described in methods. Results are mean ± SD values of at least three independent experiments. ***p<0.0001 The expression of the genes encoding for the ceramidases Ypc1p and Ydc1p also increased in Ncr1p deficient cells (Figure 9), although not so significantly as for LAG1 gene. Indeed, YPC1 and YDC1 were upregulated 5and 4-fold, respectively, in FCUP ICBAS Sphingolipid metabolism, Sit4p and TORC1 in the yeast model of Niemann-Pick type C1 disease 33 ncr1Δ cells when compared to parental cells. The deletion of SIT4 in ncr1Δ cells also suppressed these changes as β-galactosidase activity in ncr1Δsit4Δ double mutants was similar or even lower to wild type levels. Figure 9 - Expression of ceramidase genes are increased in ncr1Δ cells by a Sit4p-dependent mechanism. S. cerevisiae BY4741, ncr1Δ, sit4Δ and ncr1Δsit4Δ cells carrying LacZ reporter fusions with the promoters of YPC1 or YDC1 in the multicopy plasmid YEp357 were grown to log phase (OD600=0.6) in SC-medium lacking uracil. BGalactosidase activity was measured spectophotometrically, as described in the methods section. Results are mean ± SD values of at least three independent experiments. *p<0.05, ***p<0.0001 Taken together, these results suggest an increase in the expression of both ceramidases and ceramide synthases in ncr1Δ cells, in a Sit4p dependent manner. This indicates that sphingolipid dynamics are altered in these cells. A higher activity of these enzymes may very well be the underlying cause behind the altered sphingolipid levels in the yeast model of NPC1. 4.1.2. Modulation of sphingolipid metabolism in ncr1Δ cells The upregulation of ceramide synthase and ceramidase genes prompted us to investigate the role of these enzymes in the phenotypes presented by ncr1Δ cells. For that, we deleted those genes in Ncr1p deficient cells and assessed its impact on mitochondrial function by assessing its capacity to grow on media containing glycerol (non-fermentable carbon source). We first deleted LAG1, since the expression of this gene presented the most notorious increase in ncr1Δ cells and, therefore, Lag1p could have a major contribution to the accumulation of ceramides in this mutant (Vilaça et al. unpublished). Also, the deletion of this gene is known to increase chronological lifespan in yeast cells, which is closely related to mitochondrial function (D'mello, 34 FCUP ICBAS Sphingolipid metabolism, Sit4p and TORC1 in the yeast model of Niemann-Pick type C1 disease Childress et al. 1994). To monitor respiratory capacity, BY4741, ncr1Δ, lag1Δ and ncr1Δlag1Δ cells were grown in SC-glucose medium to exponential phase, diluted to and OD600=0.1 and fivefold dilutions were plated on SC-Glucose and SC-Glycerol plates. BY4741 and lag1Δ cells, but not ncr1Δ cells, were able to grow in glycerol plates (Figure 10). However, ncr1Δlag1Δ cells did not grown in this medium, suggesting that the deletion of LAG1 does not restore the respiratory capacity in cells lacking Ncr1p. Similar results were obtained with ncr1Δydc1Δ cells (Figure 11) as well as ncr1Δydc1Δypc1Δ triple mutants (data not shown). Figure 10 - Deletion of LAG1 in ncr1Δ cells does not restore mitochondrial function. Yeast cells were grown overnight to log phase in SC-glucose medium, then diluted to an OD600=0.1 and fivefold serial dilutions were spotted in SC-glucose or SC-glycerol plates. Figure 11 - Deletion of YDC1 in ncr1Δ cells does not restore mitochondrial function. Yeast cells were grown overnight to log phase in SC-glucose medium, then diluted to an OD600=0.1 and fivefold serial dilutions were spotted in SC-glucose or SC-glycerol plates. FCUP ICBAS Sphingolipid metabolism, Sit4p and TORC1 in the yeast model of Niemann-Pick type C1 disease 35 Myriocin is an inhibitor of serine-palmitoyl transferase, an enzyme that catalyzes the first step in the de novo synthesis of sphingolipids (Miyake, Kozutsumi et al. 1995). It was recently reported that myriocin was capable of increasing yeast chronological lifespan, through the inhibition of sphingolipid synthesis and consequent down regulation of the Pkh1/2p-Sch9p pathway (Huang, Liu et al. 2012, Liu, Huang et al. 2013). This prompted us to investigate the effect of myriocin on the mitochondrial function of ncr1Δ cells. For that, cells were grown on SC-glucose and SC-glycerol plates supplemented with 350 ng/ml myriocin. The results show that the inhibition of SPT and consequent lowering of sphingolipid burden in ncr1Δ cells was not capable of restoring mitochondrial function (Figure 12). Figure 12 - Inhibition of SPT does not improve the mitochondrial function of ncr1Δ cells. Yeast cells were grown overnight to log phase in SC-glucose medium, then diluted to an OD600=0.1 and fivefold serial dilutions were spotted in SC-glucose or SC-glycerol plates containing 350 ng/ml myriocin or equal volume of ethanol (vehicle). 36 FCUP ICBAS Sphingolipid metabolism, Sit4p and TORC1 in the yeast model of Niemann-Pick type C1 disease 4.2. Involvement of Sit4p in autophagy and Pep4 activity in the yeast model of NPC1 4.2.1. The deletion of SIT4 in ncr1Δ cells restores the autophagic flux In mammals, NPC1 deficient cells show increased basal autophagy as evidenced by the increased levels of LC3-II (Pacheco, Kunkel et al. 2007). However, there is an impaired degradation of autophagosomes (Sarkar, Carroll et al. 2013). In yeast, ncr1Δ cells also present an increased basal autophagic flux as well as an increase in the activity of the vacuolar protease Pep4p (Vilaça et al. unpublished). To investigate whether Sit4p is implicated in the modulation of autophagy in ncr1Δ cells, we analysed the processing of GFP-Atg8p in basal conditions and upon treatment with rapamycin to induce autophagy (Yorimitsu, Zaman et al. 2007). Atg8p is an ubiquitin like protein, homolog to mammalian LC3-II, and involved in the formation of autophagosomes. When autophagy is induced, GFP-Atg8p recruited to the phagophore and lipidated with phosphatidylethanolamine to drive autophagosome biogenesis (Kirisako et al., 1999). It is then delivered to the vacuole where Atg8p is degraded by resident vacuolar hydrolases. However, the GFP moiety is relatively resistant to proteolysis, whereby the appearance of free GFP signal is indicative of autophagic flux induction (Shintani and Klionsky, 2004). As previously observed, the basal autophagic flux was increased in ncr1Δ cells when compared to parental or sit4Δ cells (Figure 13). Treating cells with rapamycin was capable of inducing the autophagic flux in all strains, including ncr1Δ cells. In the double mutant ncr1Δsit4Δ, the autophagic flux was restored to levels similar to those observed for sit4Δ cells. This result implicates Sit4p in the increased autophagic flux of Ncr1p deficient cells. FCUP ICBAS Sphingolipid metabolism, Sit4p and TORC1 in the yeast model of Niemann-Pick type C1 disease 37 Figure 13 - Analysis of the autophagic flux in BY4741, ncr1Δ, sit4Δ and ncr1Δsit4 cells. Cells carrying pRS416GFP-Atg8 were grown to exponential phase in SC-Glucose medium without uracil and treated with 200 ng/ml rapamycin (Rap) or equal volume of DMSO (vehicle) for three hours. Protein extracts were analyzed by western blot using an anti GFP antibody. One out of three independent experiments is shown. 4.2.2. The deletion of SIT4 in ncr1Δ cells restores normal Pep4p activity Pep4p is the major vacuole protease in yeast (Woolford, Daniels et al. 1986). It is responsible for the posttranslational activation of percursors of vacuolar hydrolazes and degradation of cargo during autophagy. Cathepsin D, a closely related protease in mammals, is increased in NPC cells (German, Liang et al. 2002), a feature found to be conserved in the yeast model for this disease (Vilaça et al. unpublished). To assess the involvement of Sit4p in the deregulation of Pep4 in ncr1Δ cells, we measured its activity in ncr1Δ, sit4Δ and ncr1Δsit4Δ cells grown to exponential and PDS phase. In all strains, Pep4p activity increased during growth from exponential to PDS phase (Figure 14). As previously reported, ncr1Δ cells presented an increased Pep4p activity when compared to parental cells, particularly at the exponential phase (3-fold). Notably, the deletion of SIT4 in ncr1Δ cells decreased significantly Pep4p activity to the levels observed in parental and sit4Δ cells. The overall results implicate the ceramide activated protein phosphatase Sit4p in basal autophagy induction and Pep4p activation in Ncr1p deficient cells. 38 FCUP ICBAS Sphingolipid metabolism, Sit4p and TORC1 in the yeast model of Niemann-Pick type C1 disease Figure 14 - Pep4p activity is increased in ncr1Δ cells by a Sit4p-dependent mechanism. Yeast cells were grown on YPD medium to exponential (log) or post-diauxic shift (PDS) phase. Pep4p activity was measured spectrophometrically as described in methods. Results are mean ± SD values of at least three independent experiments. *p<0.05, **p<0.01, ***p<0.001 4.3. Role of Tor1p in the oxidative stress resistance and chronological lifespan of ncr1 cells. 4.3.1. TORC1 downregulation suppresses the hydrogen peroxide sensitivity of ncr1Δ cells TORC1 is a Ser/Thr protein kinase in yeast and conserved in many other eukaryotic organisms. It senses nutritional cues and regulates cellular growth as well as anabolic processes, like protein synthesis, ribosome biogenesis, among others (Loewith and Hall 2011). It also regulates transcription factors involved in the expression of stress response genes, like Gis1p and Msn2/4p, through the protein kinase Rim15p (Wanke, Pedruzzi et al. 2005, Wei, Fabrizio et al. 2008). In fact, the modulation of this pathway is known to increase longevity not only in mammals but also in yeast (Bonawitz, Chatenay-Lapointe et al. 2007, Pan, Schroeder et al. 2011, Pan and Shadel 2009). In the regulation of aging and mitochondrial function in yeast, TORC1 functions upstream of Sch9p, activating it through the phosphorylation of its Cterminal (Urban, Soulard et al. 2007). It also regulates Sit4p, by phosphorylating Tip41p/Tap42p which then interact with Sit4p and inhibit its function (Di Como and Arndt 1996). TOR is also an established inhibitor of autophagy (Wullschleger, Loewith FCUP ICBAS Sphingolipid metabolism, Sit4p and TORC1 in the yeast model of Niemann-Pick type C1 disease 39 et al. 2006, Nakatogawa, Suzuki et al. 2009), which is deregulated in ncr1Δ cells. Moreover, previous studies implicate Sch9p in the mitochondrial dysfunction, oxidative stress sensitivity and shortened lifespan of ncr1Δ cells (Vilaça et al., 2014). Since TORC1 is required for full activation of Sch9p (Urban, Soulard et al. 2007), we assessed how TORC1 downregulation affects ncr1Δ phenotypes. First, we analysed hydrogen peroxide resistance in BY4741, ncr1Δ, tor1Δ and ncr1Δtor1Δ cells, as well as in BY4741 and ncr1Δ cells treated with rapamycin for the pharmacological inhibition of TORC1. The results show that, as previously reported (Vilaça, Silva et al. 2014), ncr1Δ cells were more sensitive to hydrogen peroxide than parental cells (Figure 15). Notably, the deletion of TOR1 in ncr1Δ cells increased cellular viability (4-fold) to the levels observed in parental and tor1Δ cells. Consistently, the pharmacological inhibition of TORC1 using rapamycin had a protective effect similar to the observed upon TOR1 deletion. However, the oxidative stress resistance of ncr1Δ cells treated with rapamycin was significantly lower to that of parental cells treated with this drug. Figure 15 - Deletion of TOR1 or rapamycin treatment increases hydrogen peroxide resistance in ncr1Δ cells. (A) Cells were grown in SC-Glucose medium to earl-exponential (OD600=0.3), treated with 200 ng/ml rapamycin or equal volume of DMSO (vehicle) for three hours, and then for one hour with 1.5 mM H2O2. (B) Cells were grown to exponential phase (OD=0.6) and treated with 1.5 mM H2O2 for one hour. Results are mean ± SD values of at least three independent experiments. **p<0.01, ****p<0.0001 4.3.2. Deletion of TOR1 restores chronological lifespan in ncr1Δ cells Yeast is a well established model for studying both replicative and chronological aging (Longo and Shadel 2012). In yeast, CLS represents the time a nondividing cell survives in stationary phase. Nutrient sensing pathways, including the Tor1p-Sch9p 40 FCUP ICBAS Sphingolipid metabolism, Sit4p and TORC1 in the yeast model of Niemann-Pick type C1 disease pathway, are known to be involved in the regulation of yeast lifespan through the modulation of mitochondrial function. In fact, the downregulation of the TOR pathway in yeast has been reported to increase CLS (Bonawitz, Chatenay-Lapointe et al. 2007, Pan and Shadel 2009, Pan, Schroeder et al. 2011). Previously, it was reported that the yeast model of NPC1 presents a reduced CLS when compared to parental cells (Vilaça et al, 2014). The deletion of TOR1 in ncr1Δ cells suppresses mitochondrial dysfunction (Vilaça et al. unpublished) and the oxidative stress sensitivity (this study) of this mutant. To investigate the role of Tor1p in aging of ncr1Δ cells, we assessed the CLS of BY4741, ncr1Δ, tor1Δ and ncr1Δtor1Δ cells. Consistent with published data, CLS increased in tor1Δ cells comparing with parental cells. Importantly, the results show that TOR1 deletion also increased life span in Ncr1p deficient cells (Figure 16). In fact, the double mutant cells had a lifespan similar to the observed in tor1Δ cells and, therefore, higher to that of parental cells. This result implicate TORC1 in the premature aging of ncr1Δ cells. 0 10 20 30 40 1 10 100 tor1 ncr1to r1 BY4741 ncr1 D ays % Viable Cells Figure 16 - Deletion of TOR1 in ncr1Δ cells increases chronological lifespan. Overnight cultures were diluted to an OD600=0.6 in SC-glucose, grown for 24 hours until PDS phase and then kept in the medium overtime. Cellular viability was expressed as the percentage of colony forming units, in relation to t0. Results are mean ± SD values of at least three independent experiments. FCUP ICBAS Sphingolipid metabolism, Sit4p and TORC1 in the yeast model of Niemann-Pick type C1 disease 41 CHAPTER 5 DISCUSSION 48 FCUP ICBAS Sphingolipid metabolism, Sit4p and TORC1 in the yeast model of Niemann-Pick type C1 disease Almeida, T., M. Marques, D., Mojzita, M. A. Amorim, R. D. Silva, B. Almeida, P. Rodrigues, P. Ludovico, S. Hohmann, P. Moradas-Ferreira, M. Corte-Real and V. Costa (2008). 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