Autophagy dysfunctions and lysosomal permeabilization in Tay-Sachs and Sandhoff diseases

Autophagy dysfunctions and lysosomal permeabilization in Tay-Sachs and Sandhoff diseases TESIS DOCTORAL BEATRIZ CASTEJÓN VEGA SEVILLA, 2020 1 2 Autophagy dysfunctions and lysosomal permeabilization in Tay-Sachs and Sandhoff diseases TESIS DOCTORAL BEATRIZ CASTEJÓN VEGA SEVILLA, 2020 3 4 Autophagy dysfunctions and lysosomal permeabilization in Tay-Sachs and Sandhoff diseases Tesis Doctoral Memoria de tesis doctoral presentada por BEATRIZ CASTEJÓN VEGA, graduada en Bioquímica por la Universidad de Sevilla, para optar al título de Doctor enmarcado en el programa de Doctorado en Ciencias de la Salud. Esta tesis doctoral ha sido realizada en la Universidad de Sevilla desde el año 2016 hasta el año 2019. Esta tesis está inscrita al Departamento de Estomatología de la Facultad de Odontología de la Universidad de Sevilla. BEATRIZ CASTEJÓN VEGA SEVILLA, 2020 5 6 7 8 “Science and everyday life cannot and should not be separated “ Rosalind Franklin 15 16 Contents Abbreviations……………………………………………………………………………….19 Introduction ..................................................................................................................... 29 1. Rare diseases ...................................................................................................... 29 2. Lysosomal storage diseases ............................................................................... 37 Causes of LSDs ...................................................................................................... 43 Symptoms................................................................................................................ 47 3. Gangliosides ........................................................................................................ 50 4. Tay-Sachs and Sandhoff diseases ..................................................................... 53 Symptoms................................................................................................................ 58 Treatments .............................................................................................................. 60 Genetic mutations ................................................................................................... 61 5. mTOR Pathway ................................................................................................... 65 Upstream signalling ................................................................................................. 65 Downstream signalling ............................................................................................ 69 6. Autophagy ............................................................................................................ 71 Autophagy dysfunction ............................................................................................ 74 7. Mitochondria ........................................................................................................ 77 AIMS ............................................................................................................................... 81 Material and methods ..................................................................................................... 85 Reagents ..................................................................................................................... 85 Ethical statements ....................................................................................................... 87 1. Cell culture ........................................................................................................... 87 2. Mouse model ....................................................................................................... 88 3. Structural mutation analysis ................................................................................ 88 4. Enzymatic activity of HexA .................................................................................. 88 5. Proliferation assay ............................................................................................... 88 6. Treatments ........................................................................................................... 89 7. Immunoblotting assay ............................................................................................. 89 8. Monitoring the autophagosome formation .......................................................... 90 9. Immunofluorescence Microscopy ........................................................................ 91 10. Electron Microscopy Imaging .......................................................................... 91 11. Cathepsin B and HexA release ....................................................................... 92 12. Galectin 3 puncta ............................................................................................. 92 17 13. Cell fractionation .............................................................................................. 93 14. Puromycin assay .............................................................................................. 93 15. CRISPR/CAS9 ................................................................................................. 93 16. Measurement of mitochondrial generation of superoxide ............................... 97 17. Lipid peroxidation ............................................................................................. 97 18. Antioxidant enzyme activity ............................................................................. 97 19. Lipid hydroperoxides ........................................................................................ 98 20. Measurement of citrate synthase activity ........................................................ 98 21. Analysis of mitochondrial network and morphology ........................................ 98 22. Mitophagy analysis .......................................................................................... 98 23. TMRM assay .................................................................................................... 99 24. Analysis of mitochondrial respiratory function using Seahorse extracellular flux analyser ....................................................................................................................... 99 25. ATP levels ...................................................................................................... 101 26. Microarrays .................................................................................................... 101 27. Microscopy ..................................................................................................... 103 28. Statistical analysis .......................................................................................... 104 Results .......................................................................................................................... 108 1. Characterization of Tay-Sachs and Sandhoff patients ..................................... 108 2. Autophagic flux is impaired in fibroblast from Tay-Sachs and Sandhoff patients 119 3. Lysosomal permeabilization is associated with Tay-Sachs and Sandhoff diseases .................................................................................................................... 136 4. mTOR pathway is downregulated ..................................................................... 149 5. Mitochondrial dysfunction and oxidative stress are associated with Tay-Sachs and Sandhoff diseases ............................................................................................. 163 6. L-Arginine treatment recovers mTOR activity and lysosomal dysfunction ....... 183 7. Transcriptome analysis reveal altered molecular pathways ............................. 208 Discussion .................................................................................................................... 229 Conclusions .................................................................................................................. 239 References ................................................................................................................... 242 Appendix ....................................................................................................................... 257 1. Publications ....................................................................................................... 257 2. Mettings ............................................................................................................. 259 Agradecimientos ........................................................................................................... 263 18 19 ABBREVIATIONS 20 Abbreviations 3D: Three-dimensional ADP: Adenosine diphosphate AKT: Protein kinase B AL: Albumin AMP: Adenosine monophosphate AMPK: 5ʹ-adenosine monophosphate activated protein kinase APS: Ammonium persulfate Arg: Arginine ATG: Autophagy-related ATP: Adenosine triphosphate BafA1: bafilomycin A1 Bp: Base pair BSA: Bovine serum albumin cAMP: Cyclic AMP Cas9: CRISPR-associated protein 9 CatB: cathepsin B CCCP: Carbonyl cyanide mchlorophenylhydrazone CMA: Chaperone-mediated autophagy CNS: central nervous system CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats CSF: Cerebral spinal fluid CytC: Cytochrome c 21 DAPI: 4′,6-diamidino-2-phenylindole DMEM: Dulbecco's modified Eagle's medium DMSO: Dimethyl sulfoxide DNA: Deoxyribonucleic acid ECAR: Extracellular acidification rate EDTA: Ethylenediaminetetraacetic acid EM: electron microscopy ER: Endoplasmic reticulum ETC: Electron transport chain FACS: Fluorescence-Activated Cell Sorting FBS: Fetal bovine serum FCCP: Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone FDA: Food and Drug Administration FITC: Fluorescein Isotiocyanate FOR: Forward primer GAPDH: Glyceraldehyde-3-phosphate dehydrogenase GFP: Green fluorescent protein GM2A: GM2 ganglioside activator GO: Gene Ontology GTP: Guanine triphosphate HBSS: Hank's balanced salt solution HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HEXA: gene encoding α-subunit of β-hexosaminidase A 22 HEXB: gene encoding β-subunit of β-hexosaminidase A HIF1: Hypoxia-inducing factor-1 HPO: human phenotype ontology HRP: Horseradish peroxidase IMM: Inner mitochondrial membrane IS: Intermembrane space LC3: Microtubule-associated proteins 1A/1B light chain 3 LC3B: LC3 isoform (antibody against LC3B was used during this thesis) LC3-I: LC3 cleaved at the C terminus by Atg4 LC3-II: LC3-I lipided by conjugation to PE LDH: lactate dehydrogenase Leu: Leucine LMP: lysosomal membrane permeabilization LROs: lysosome related organelles LSDs: lysosomal storage diseases M6P: mannose-6-phosphate MFN1: Mitofusin-1 MFN2: Mitofusin-2 MMPs: Matrix metalloproteinases MnSOD: Superoxide dismutase MPP: Mitochondrial processing peptidase MPT: Mitochondrial permeability transition MRC: Mitochondrial respiratory chain mRNAs: Messenger RNAs 23 mt-aaRSs: Aminoacyl mt-RNA synthetases mtDNA: Mitochondrial DNA MTDR: MitoTrackerTM Deep Red mTOR: Mammalian target of rapamycin mTORC1: mTOR complex 1 mTORC2: mTOR complex 2 mt-rRNAs: Mitochondrial ribosomal RNAs mt-tRNA: Mitochondrial transfer RNA NADH: Reduced nicotinamide adenine dinucleotide nDNA: Nuclear DNA NGS: next generation sequence NTs: nucleotides O/N: Overnight OCR: Oxygen consumption rate OMM: Outer mitochondrial membrane OPA1: Optic atrophy 1 P: P-value (statistics) P62/SQSTM1: Sequestosome-1 p70S6K: Ribosomal S6 kinase PAGE: Polyacrylamide gel electrophoresis PAS: Phagophore assembly sites PBS: Phosphate-buffered saline PCR: Polymerase chain reaction PE: Phosphatidylethanolamine PFA: Paraformaldehyde Pi: Inorganic phosphate 24 PI3K: Phosphatidylinositol kinase type III PI3K: phosphoinositide 3-kinase PI3P: Phosphatidylinositol 3-phosphate PINK1: PTEN-induced putative kinase 1 PKB: Protein kinase B PRAS40: Proline-rich Akt substrate of 40 kDa RE: Restriction enzyme RHEB: Ras homolog enriched in brain RIPA: Radioimmunoprecipitation assay (buffer), 50 mM Tris-HCl, 150mM sodium chloride, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1%SDS, pH 8.0. RNA: Ribonucleic acid ROS: Reactive oxygen species REV: Reverse primer rRNAs: Ribosomal RNAs RT: Room temperature SAP: sphingolipid activator proteins SD: Sandhoff disease SD: Standard deviation SDH: Succinate dehydrogenase SDS: Sodium dodecyl sulphate sgRNA: Single guide RNA shRNA: Short hairpin RNA TBS: Tris-buffered saline TEMED: Tetramethylethylenediamine TF: transcription factor TG: Transfer buffer (TGS: Running buffer (25 mM Tris, 190mM glycine, 0.1% SDS, pH 8.3) 31 Table 1| Prevalence criteria for the definition of Rare Diseases in selected countries. (Field & Boat, 2010). It is known that individual RDs affect only a few people but if they are studied as a whole it creates an important health problem worldwide. It is documented that RDs impact more people than cancer and AIDS combined (“RARE Facts - Global Genes,” n.d.), (Dharssi, Wong-Rieger, Harold, & Terry, 2017). In the following graphs (Figure 2 and 3) it is shown the number of RDs and their prevalence per 100.000 and the number of individual and family cases obtained from Orphanet 2019. It is very significative the increasing amount of RDs with very low prevalence, showing the variability and variety of these disorders. Country Prevalence Criterion Prevalence expressed as Rate for Year of Policy Adoption United States 200.000 people 1984: 85/100.000 2008: 66/100.000 Australia 2000 people 1998: 11/100.000 2008: 9/100.000 European Union 5/10.000 population (aprox. 250.000 people in 27 EU nations) Not applicable Japan 50000 people 1993: 40/100.000 2088: 39/ 100.000 32 Figure 2| Number of rare diseases by prevalence. A) Prevalence up to 50/100.000. B) Prevalence per 10/10.000 or less. Source: Orphanet, 2019. Figure 3|Number of rare diseases by A) number of individual cases and B) number of family cases. Source: Orphanet, 2019. 33 These disorders, 65% of them are disabling, are present throughout the whole life of the person and usually involving lots of pain to the patients and their families. They can be acute, chronic and degenerative and affect people of all ages, although around 75% affects only children, which may die before the age of five years old. The earlier the symptoms appear the worst the prognosis and the patients` life are usually compromised with a higher risk of early death (Field & Boat, 2011). They are characterized by a wide range of disorders and symptoms which usually vary from disease to disease, even among patients suffering from the same disease, making the misdiagnosis highly frequent (“About Rare Diseases | www.eurordis.org,” n.d.). It is recorded that different pathogenic variants of the same gene may have different effects. These should be considered different diseases because their symptoms and the treatments are different (Haendel et al., 2019). Most rare diseases are undiagnosed for years while physicians do their best to understand a condition that they have never seen before. Due to the lack of recognizable sings, there are changes in the diagnosis when new or advancing symptoms appear. The diagnosed process is the most feared by the patients and families, who have to visit different doctors before been diagnosed. Since the first symptomatology appears until the disease is diagnosed it easily takes around 5 years, some of them even 10 or more years. They feel desolate as they have to face the challenges of finding information about their diseases and specialists who help them in the diagnosis and subsequent treatment. Patients, relatives and friends, in their finding for answers, usually join or create organizations to support other families and enhance the visibility of the diseases. There is a growing societal pressure to dedicate more investment in the research of these conditions and make translational the findings making new medicaments and faster diagnosis. Apart from the diagnosis, the lack of treatments is other main problem that people with rare disease have to deal with. It´s highly common these pathologies haven’t got a treatment to cure or weaken the diseases and if they have it, it is not effective. The 26,8% of patients have received an unappropriated treatment during their lives. In 1983 in United States the Orphan Drug Act took place to encourage the development of drugs for rare diseases. 34 Before these date there isn’t enough motivation in the development of orphan drugs, however three decades later, there were a high proportion of industry researching and developing new drugs (Herder, 2017). Due to the reduce amount of affected people, in comparison with others more common diseases, pharmaceutics industries don’t see the profit in investing in the research in the molecular mechanisms that underlying these pathologies and translate the advances into potential medicines, known as orphan drugs. They provide little financial incentive to produce new treatments because there aren´t enough patients to make research cost-effective. In addition, the fact that there aren’t enough patients is a challenge to undertake the clinical trials. Researchers required accurate statistical analysis to measure the drug effects and compare the results between treatment and placebo (Gavin, 2015). In United States the orphan drug designation should be obtain through the Office Orphan Products Development of the FDA showing that the diseases prevalence is less than 200.000 in USA. On the other hand, in the European Union the use of orphan drugs should be evaluated by the Committee for Orphan Medicinal Products of The European Medicines Agency. The prevalence should be less than 5 in 10.000 in EU, approximately 254.000 people (Tambuyzer et al., 2019). Nowadays it is estimated a number of 7.000 rare diseases and new ones are discovered each year, most of them haven´t got an approved treatment. Lots of effort is being done to elucidate the molecular mechanisms underlying rare diseases by a huge number of researchers worldwide. However, the rate of translation the knowledge acquired into therapies and treatments is too low. The limitation in the knowledge is the main obstacle for improving the therapies, nonetheless as the Figure 4 shows the number of science articles about these disorders is continuously growing but production of new treatments is still a challenge. 35 Figure 4| Translational gap between the production of science articles and orphan drug approvals. The graph shows the number of paper published about RDs per year from 1987 to 2018 and the number of orphan drugs approved by the FDA and the European Medicines Agency (EMA) on a logarithmic scale. Graph taken from (Tambuyzer et al., 2019). Diverse therapeutics modalities are being tried to make translational the scientific advances in the understanding of RDs. Pharmaceutics industry and researchers used to focus on small-molecule drugs which are the best stablished, low production costs, multiple routes of administration and controlled dosing. However, due to the new advances in molecular biology and understanding the human genome through genome sequencing new drugs are being discovered. Such as protein-based therapeutics (proteins, peptides and antibodies), antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs) and gene and cell therapies. The difference between these modalities is the way they target the pathways implicated in these disorders and the ability to reach cellular compartments. AOSs, siRNA and gene al cell therapy works in deeper levels regulating gene transcription and protein production, however protein-based therapeutics balanced and modulate the extracellular targets, 36 replace dysfunctional proteins and enhance the lack of certain proteins (Tambuyzer et al., 2019). Nowadays, because of the high interest in rare diseases knowledge and the aim for improve the diagnosis and develop effective new treatments new technology is used. Next-generation sequencing (NGS) is being used to identify genes which cause rare diseases and The Human Phenotype Ontology (HPO) is intended to analysis the Human Phenom. Standardization of a disease is a must and integrating phenotype based database and genetic information is essential to reach our goals (Jia et al., 2017). 37 2. Lysosomal storage diseases Lysosomal storage diseases (LSDs) are a heterogeneous group of around 70 individually rare genetic diseases (Table 2) which are inherit, autosomal recessive (except for the three X-linked disorders which are Fabry, Hunter and Danon diseases (Platt,F.M. et al, 2018), (Pastores & Wang, 2017)) and are characterized by the accumulation of un-degraded cellular products inside the cells. Patients who are suffering from these disorders can be homozygous for the same mutation in both alleles or heterozygous for two different mutations. In other words, heteroallelic patient having one mutation in one allele and a different one in the other allele (Haskins & Giger, 2008). The age of the onset and the clinical signs vary between patients and the severity of the mutation. The amount of residual activity of the enzyme influences the age of the symptoms onset. Infantile type is the most severe and frequent corresponding to a null or almost null enzymatic activity, following by late-infantile, juvenile and adult type (Vitner, Platt, & Futerman, 2010). Individually these diseases represent a very tiny part of the population with an estimated incidence of 1 in 5000-5.500; however as a whole they are common with an estimated incidence of 1-50.000-250.000 live births. Among LSDs the most common is Fabry disease (up to 2,5 cases per 100.000 males), Gaucher disease and Pompe disease (2 cases per 100.000 individuals). The estimated prevalence as a group is 1 in 8000 live births. Nevertheless, many of the LSDs are individually common in specific ethnicities, for instance, Gaucher disease type I, Tay-Sachs disease and Niemann-Pick disease type A are very common within the Ashkenazi Jews (100, 25 and 2,5 per 100.000 individuals respectively); Metachromatic Leuckodystrophy in Western Navajo Nation (40 per 10.000 individuals): Fabryc disease in Nova Scotia (9,3 per 100.000 individuals) and Pompe disease in the Netherlands (2,5 per 100.000 individuals) (“Lysosomal storage diseases | Nature Reviews Disease Primers,” n.d.), (Parenti, Andria, & Ballabio, 2015). 38 Stored Substrate Disease Protein deficiency Gene locus Lipids Sulfatides Metachromatic leukodystrophy Arylsulfatase A (galactose-3-sulfatase) ARSA, 22q13.33 GM1 gangliosides, sphingomyelin, glycolipids, sulfatide Prosaposin deficiency(Metachrom atic leukodystrophy variant) Prosaposin( Saposin B activator) PSAP, 10q22.1 Glycolipids, gangliosides, phospholipids Sialolipidosis (Mucolipidosis IV, Berman disease) Galactosialidosis Mucolipin-1 Protective protein/cathepsin A MCOLN1, 19p13.2 CTSA, 20q13.12 Glucosylceramide, globosides Gaucher disease (three types)* β-Glucosidase GBA, 1q22 Glucosylceramide, globosides Saposin C deficiency (Gaucher disease variant) Saposin B deficency Saposin C Saposin B PSAP, 10q22 PSAP 10q22.1 Ceramide Farber disease (seven types) Acid ceramidase ASAH1, 8p22 Sphingomyelin Niemann–Pick disease types A and B Sphingomyelinase SMPD1,11p15. 4 Cholesterol esters Wolman disease, cholesteryl ester storage disease Lysosomal acid lipase LIPA, 10q23.2- q23.3 Cholesterol, sphingomyelin Niemann–Pick disease type C Cerebrotendinous xanthomatosis NPC1; NPC2 Sterol 27-hydroxylase NPC1, 18q11.2 NPC2, 14q24.3 CYP27A1, 2q33-qter 39 Lipofuscin Neuronal ceroid lipofuscinosis (many types)** PPT PPT1 1p32 (Inf/Juv) Lipids and Olygosacharides GM2 gangliosides, glycolipids, globoside oligosaccharides Tay-Sachs disease (GM2 gangliosidosis) (three types) Sandhoff disease (GM2 gangliosidosis) GM2 gangliosides AB variant Α Subunit of βhexosaminidase β Subunit of βhexosaminidase GM2 activator HEXA, 15q23 HEXB, 5q13.3 GM2A, 5q33.1 GM1 gangliosides, oligosaccharides, keratin sulphate, glycolipids GM1 gangliosidosis (three types) β-D-galactosidase GLB1, 3p22.3 Galactosylceramides Krabbe disease Galactocerebrosidase GALC, 14q31.3 Oligosaccharides, glycosaminoglycans, glycosphingolipids Mucolipolisis IIIC γ N-acetylglucosamine-1- phosphotransferase subunit γ GNPTG, 16p13.3 Oligosaccharides, glycosaminoglycans, glycosphingolipids I-cell (ML-II) N-acetylglucosamine-1- phosphotransferase subunits α/β GNPTAB, 12q23.2 * Glycolipids, oligosaccharides Mucolipidosis III A/B(pseudo-Hurler polydystrophy) N-acetylglucosamine-1- phosphotransferase subunits α/β GNPTAB, 12q23.2 α-Galactosylsphingolipids, oligosaccharides Fabry disease α-Galactosidase A GLA, Xq22.1 Sulfatides, glycolipids, glycosaminoglycans Mucosulfatidosis (Multiple sulfatase deficiency) Sulfatase-modifying factor 1(SUMF-1) SUMF1, p26.1 Fucosides, glycolipids α-Fucosidosis α-Fucosidase FUCA1, 1p36.1 40 Oligosaccharides Glycogen Pompe disease Danon disease Acid α-D-glucosidase Lysosomal associated membrane protein-2 (LAMP-2) GAA, 17q25.3 LAMP2, Xq24 α-Mannoside α-Mannosidosis α-Mannosidase MAN2B1, 19p13.2 β-Mannoside β-Mannosidosis β-Mannosidase MANBA, 4q24 N-acetylgalactosaminide Schindler (Type I, infantile severe form) Type III (Intermediate form) Kanzaki disease (Type II, adult onset form) α-N- acetylgalactosaminidase NAGA, 22q13.2 Sialyloligosaccharides Sialidosis I-II α-Neuraminidase (sialidase) NEU1, 6p21.33 Sialyloligosaccharides, galactosyloligosaccharides Galactosialidosis (Goldberg syndrome, combined neuraminidase and B- galactosidase deficiency) Winchester syndrome Protective protein/ cathepsin A ( βgalactosidase, αneurominidase) Metalloproteinase-2 PPCA, 20q13.12 GLB1, 3p22.3 NEU1, 6p21,33 MMP2, 16q13 Sialic acid, glucuronic acid Salla disease( Infantile free sialic acid storage disease) Sialin (lysosomal transporter) SLC17A5, 6q13 Free acid sialic acid Sialuria (French type) UDP-N- acetylglucosamine-2- epimerase/N- acetylmannosamine kinase GNE, 9p12- p11 47 Table 3| Different mechanisms of Lysosomal Storage Disorders. Data were taken from (Alroy & Lyons, n.d.). NPC, Niemann-Pick C. Symptoms The symptomatology of these diseases is very wide and different between patients, even with the same disease. Although lysosomal and lysosomalrelated genes are expressed in all the cells of the body, the non-degraded substrates are stored in the tissues in which these substrates are usually turned-over (Pastores & Wang, 2017). Among their clinical manifestations such as visceral (hepatosplenomegaly), haematological (enlarged, vacuoles full of non-degraded material visible in lymphocytes or histiocytes), ocular (corneal or lenticular opacities, retinal abnormalities such as cherry red spot and retinal dystrophy, glaucoma, optic nerve atrophy and blindness) and skeletal (bone dysplasia, joints limitations, anomaly in bone density, osteonecrosis, areas of bone infarction) the neurological ones are the most recurrent, being a common hallmark of LSDs (comprises two-thirds of all lysosomal diseases). As it is shown in the Figure 6, these diseases may affect any body organ or system, including the heart (valvular thickening, cardiomegaly, cardiac failure and arrhythmias), skin (hypohidrosis, angiokeratoma), lungs, intestine, upper respiratory tract and kidneys. The phenotype between LSDs is commonly overlapping because different diseases causing mutations affect the same organ or system (Parenti et al., 2015) As rare diseases, the diagnosis of the Failure in the fusion between endosome or lysosome due to mutation on the lysosomal membrane protein 2 gene (LAMP-2) Danon disease Decrease in transport of degradation end products out of lysosomes Salla disease, infantile sialidosis, and cystinosis 48 disorder is very confusing and requires a combination of morphological, clinical, biochemical and molecular biology techniques. 49 Figure 6|Organs affected in LSDs and LROs. Mutations causing LSDs and LROs are responsible for a huge range of symptoms which affect different organs. Although, it is documented that these diseases deeply affect the brain, it is very frequent that they involve multiple organs systems. Blue boxes show the mutations that causes specific clinical manifestations and green ones contain mutations that cause non-specific symptoms. The diseases caused by each mutation are shown in the Table 2. Figure data were taken from (“Lysosomal storage diseases | Nature Reviews Disease Primers,” n.d.). 50 3. Gangliosides Gangliosides are complex glycolipids formed by a ceramide (sphingosine and stearic acid - hydrophobic lipid portion) linked to a chain of a variable number of monosaccharides with a different number of sialic acid residues (hydrophilic sugar portion). These components make an acidic and negatively charge molecule at pH 7. Gangliosides are synthetized in the endoplasmic reticulum and then they are modified by adding different number of carbohydrates in Golgi apparatus. They are characterized by their high level of heterogeneity and diversity of the carbohydrates chain, making a total amount of 188 different gangliosides in vertebrates. They are also known as glycosphingolipids which arise from the link between a ceramide and an oligosaccharide. There are four different types of glycosphingolipids: cerebrosides, sulfatides, globosides and gangliosides. However, above all, gangliosides are the most abundant in the nervous system suggesting their functions in its development and function. They are also distributed in all body cell and tissues. Primarily gangliosides are localized in the outer layer of cell membrane along with sphingomyelin, cholesterol, signalling and adhesion proteins in lipids rafts. The hydrophilic sugar portion with the sialic acid is extended from the plasma membrane while the hydrophobic tails are anchored in the membrane. They are essential components of cell membranes and are involved in cell-cell recognition, adhesion and signal transduction. They are also found in nuclear membrane and it has been hypothesized to be involved in the maintenance of intracellular and intranuclear calcium homeostasis (Yu, Tsai, Ariga, & Yanagisawa, 2011), (Furukawa et al., 2018). The gangliosides are named according to the Svennerhom nomenclature, providing information about its structure: G refers to ganglioside; the following letter A, M, D or T indicates the number of sialic acid molecules (zero, one, two and three respectively) and the number 1, 2 or 3 designates the number of monosaccharides residues (four, three, and two respectively) (Patterson, 2013), (“Ganglioside GM2 (d18:1/25:0) | C74H135N3O26 - PubChem,” n.d.). 51 Inherit defects in the enzymes involve in gangliosides catabolism are the responsible for many LSDs such as GM1 and GM2 gangliosidoses (lysosomal accumulation of GM2 gangliosides, Figure 7). Many studies have been done with genetically engineered mice deficient in ganglioside synthase showing the essential and critical role of gangliosides. It has been documented that GM3 gene KO in mice result of complete deafness and attention-deficit hyperactivity disorder, suggesting the role of gangliosides in the maturation of the cochlea and the maintenance of neuropsychological balance (Yoshikawa et al., 2009), (Niimi et al., 2011). Mice lacking GM2 synthase show impaired motor coordination and lower nerve conduction velocity with a decrease in myelination in sciatic and optic nerves with neural degeneration; suggesting the importance of these molecules in neural functions and structural maintenance of nervous system (Takamiya et al., 1996), (Sugiura et al., 2005). In addition, double KO mice of two gangliosides synthases (GM2 and GM3) show severe lethality. However, it still remains unknown if these neurological abnormalities are caused by the lack of these enzymes or because of the substrate precursors accumulation (Yu et al., 2011). 52 Figure 7| Structure of GM2 Ganglioside. GM2 ganglioside is a glycosphingolipid formed by a ceramide (steraric acid and shingosine) linked with a oligosaccharide (β-D-glucose, β-D-galactose and N-acetyl- β-D- galactosamine) and one molecule of sialic acid. 53 4. Tay-Sachs and Sandhoff diseases Tay-Sachs (TSD, OMIM 272800) and Sandhoff (SD, OMIM 268800) are two rare inherit LSDs which affects the nervous system and are characterized by early neuronal cell death and progressive neurodegeneration due to an intraneuronal accumulation of incompletely metabolized GM2. It is known that there is an increase of 100 to 300 times of the brain levels of GM2 accumulation in these diseases compared with a healthy person. These autosomal recessive diseases belong to a group of disorders named as GM2 gangliosidoses, which are disorders that result of the failure of GM2 degradation leading to cell death. They are cause by the deficiency of the enzyme involves in their degradation, β- Hexosaminidase A (HEXA, E.C. 3.2.1.52). The severity and progression of the diseases depend on the activity level of the enzyme (residual activity) due to the gene mutations. It means that the time of onset and the symptoms reflects the degree of enzymatic dysfunction. As another lysosomal glycosidase, HexA can hydrolyse numerous substrates such as glycoproteins, oligosaccharides, glycosaminoglycans and glycolipids which include GM2 gangliosides. It cut specifically the β-linkage of the terminal non-reducing sugar of the substrate (N- acetyl glucosamine or N-acetyl galactosamine) (Schuchman & Simonaro, 2013). In Tay-Sachs (variant B) there are mutations in the HEXA gene located on the long arm of the chromosome 15 (15q23-q24) which encodes for the α-subunit of HexA. This chromosomal region is approximately 35kb in length and contains 14 exons. In Sandhoff (variant O) there are mutations on the HEXB gene located on the long arm of the chromosome 5 (5q13) that is approximately 45kb in length and is divided in 14 exons, which encodes for the β-subunit of the HexA. Both genes share a common exon structure and their promoter activity resides within approximately 150bp of the first ATG codon. In addition, both genes are evolutionary related and their subunits share approximately 57% amino acid identity (Schuchman & Simonaro, 2013). Homodimers and heterodimers formed by these subunits lead to the three Hexosaminidase isozymes in mammals: Hexosaminidase S is a homodimer formed by two αsubunits (αα), Hexosaminidase A is a heterodimer formed by one α-subunit (αβ) 54 and one β-subunit and Hexosaminidase B is a homodimer composed by two βsubunits (ββ). The most extensive forms are HexA and HexB, however only HexA is able to degrade the GM2 gangliosides. There is also another GM2 variant which is an extremely rare genetic disease called as AB variant (OMIM 272750) which is caused by mutations in the GM2A gene on chromosome 5 (5q31.1-31.3), which is approximately 16kb containing 4 exons. This gene encodes for GM2 activator protein (GM2A) that it is essential in the degradation of GM2 gangliosides because it acts like a cofactor for the HexA enzyme (Ferreira & Gahl, 2017), (Schuchman & Simonaro, 2013). Consequently, mutations in any of these 3 genes may lead to GM2 gangliosides with very similar pathology except for the visceral organ involvement of Sandhoff patients. In fact, there are three different types of gangliosidoses all of them characterized by the GM2 gangliosides as the primary neuronal storage material: Tay-Sachs with normal levels of HexB activity but deficient activity of HexA; Sandhoff with deficient levels of both enzymes HexA and HexB and GM2 activator deficiency with normal levels of HexA and HexB but with the inability to form a proper GM2-GM2A complex (Figure 8) (Ferreira & Gahl, 2017). 55 Figure 8|GM2 gangliosidoses variants. GM2 gangliosidoses are a group of LSDs which are characterized by the lysosomal accumulation of non-degraded GM2 gangliosides which leads to cell death. There are three variants of GM2 gangliosidoses: B, O and AB caused by mutation in different genes HEXA, HEXB and GM2A, respectively. The most common is Tay-Sachs disease (B Variant) caused by a mutation in HEXA gene encoding for the α-subunit; Sandhoff (O Variant) caused by a mutation in the HEXB gene which encodes for the β-subunit and GM2A (AB Variant) caused by a mutation in GM2A gene which encodes for a GM2 activator protein. Both subunits form the three mammal’s isoforms of the β-hexosminidase: HexS (αα), HexA (αβ) and HexB (αβ). The role of HexA and GM2A are essential for the correct hydrolysis of the GM2 gangliosides, since HexA is the unique enzyme which is able to degrade these substrates and GM2A acts as a cofactor for the enzyme forming a 56 complex with GM2 ganglioside and presenting to the HexA inside the lysosomes. GM2 gangliosides are a type of gangliosides which is not completely metabolized in Tay-Sachs and Sandhoff diseases. They are primarily an intermediate in the degradation and synthesis of higher brain gangliosides such as GM1 (Figure 5). The letter G refers to ganglioside, M indicates the number of sialic acid molecules and the number 2 refers to the carbohydrate sequence that is attached to the ceramide (Figure 7). GM2 gangliosides are primarily located in the outer leaflet of the plasma membrane and are catalysed in lysosomes (acidic cellular organelles) by the HexA. The substrate specify of both β-hexosaminidase enzymes (HexA and HexB) depends on the charge of the binding pockets of their subunits and on the mechanism by which the GM2-GM2A complex interacts with the HexA. Both enzymes can hydrolyse soluble and neutral substrates but only HexA, because of its α-subunit is positively charge (αAsn423 and αArg424), can catabolize negatively charge GM2 gangliosides (the negative charge is given by the sialic acid molecule). On the contrary, the β-subunit of HexB is negatively charge (βAsp452 and βLeu453) and repels the GM2 ganglioside (Tropak et al., 2016). HexA is formed by two subunits (α and β), both of them have an active site but the dimerization is essential for its activity. Whereas β subunit prefers neutral and water-soluble substrates, α subunit also hydrolyse negatively charge molecules such as GM2 gangliosides (Schuchman & Simonaro, 2013). Both HexA subunits have their own active site, but their dimerization is essential for the enzyme activity due to the stabilization given by the residues in the active site surroundings. Thus, monomers by themselves are not active (Ferreira & Gahl, 2017), (Dersh, Iwamoto, & Argon, 2016), (Tropak et al., 2016). HexA are synthesized, glycosylated, intramolecular disulphuric bonds formed and dimerize in the ER where GM2A is synthesized too and then, both of them are exposed to subsequent alteration such as modification of specific glycan with mannose-6-phosphate (M6P) in Golgi apparatus. Then, both of them are transported by the mannose 6 phosphate receptor to the inside of the lysosomes where they are proteolytically processed to their active form. GM2A 63 Figure 12|Characteristic of HEXB pathogenic mutations. A) Types of mutations, B) mutations consequences and C) gene area where the mutation is located. (“Graphs & statistics on gene HEXA - Global Variome shared LOVD,” n.d.). It has been identified around 74 HEXA mutations and 20 HEXB mutations along their genes. It is interesting that in the exon 5 of HEXA gene there is a large cluster of mutations and in the same exon in HEXB gene there is only one mutation. In addition, in HEXB gene it has been found two big deletions of 16 and 50 kb and in HEXA there are only tiny ones (Figure 13), (Triggs-Raine et al., 2001). 64 Figure 13|Simplified representation of the A) HEXA and B) HEXB gene mutations described. The horizontal line represents the gene; black boxes represent the 14 exons of the gene. Figure was taken from (Triggs-Raine et al., 2001). 65 5. mTOR Pathway The mammalian mechanistic target of rapamycin (mTOR) is a 289-kDa serinethreonine kinase that belongs to the phosphoinositide 3-kinase (PI3K)-related kinase (PIKK) family and is considered the master regulator of cellular metabolism. It regulates metabolism, cellular growth, protein synthesis, tissue repair and regeneration, cytoskeletal organization, cellular survival, aging and stem cell regeneration or quiescence. This high-molecular weight protein is formed by 2549 amino acids and is encoded by a single gene in the chromosome 1 (1p36.22). It is the catalytic subunit of two major protein complexes, each one with different kinase specificity and thus, different downstream signalling to control cellular functions; mTORC1 with six proteins and mtorC2 with seven proteins, which are involved in different roles. mTORC1 is formed by mTOR, regulatory-associated protein of mTOR (RAPTOR), mammalian lethal with SEC13 protein 8 (Mlst8, also GβL), DEPTOR, TTI1/TEL2 and proline-rich protein kinase C substrate (PRAS40); while mTORC2 are composed by mTOR, the rapamycin-insensitive companion of mTOR (RICTOR), mammalian stress-activated protein kinase interacting protein 1 (mSIN1), Proctor1/2, DEPTOR and TTI1/TEL2 (Ainslie, Gibson, & Vogel, 2016), (Kim & Guan, 2015), (Cuyàs, Corominas-Faja, Joven, & Menendez, 2014). Deregulation of mTOR signalling has been implicated in the onset and development of many human diseases such as neurodegenerative diseases, cancer and diabetes (Kim & Guan, 2015). Upstream signalling Growth factors act upstream mTORC1 binding with their specific tyrosine kinase receptor (RTKs) leading to the activation of several kinases such as AKT, extracellular signal-regulated kinase (ERK) and ribosomal S6 kinase (RSK). All of them finally converge into the tuberous sclerosis complex 1 and 2 (TSC1 and TSC2) signalling pathway (Paquette, El-Houjeiri, & Pause, 2018). One of the best studied upstream regulators of mTORC1 is the growth factor/PIK/AKT pathway. Different growth factors can act on their specific RTKs to induce intracellular phosphorylation events leading to the recruitment of PI3K 66 to the plasma membrane. The PI3´-OH Kinase (PI3K) generate PI-3,4,5- triphosphate (PIP3) from PI-4,5 bisphosphate (PIP2) due to its activation by RTKs or by G protein-coupled receptors (GPCRs). PIP2 acts as a second messenger by biding to and activating PH domain-containing proteins such as Akt which is recruited to the plasma membrane as well. Akt is activated by the phosphorylation at S308 by PDK1 kinase and by mTORC2 at S473 residue; the mTORC2 Akt phosphorylation is critical for its maximal activity. On the contrary, PIP3 is hydrolysed by PTEN forming PIP2. Then, Akt phosphorylates several downstream targets and generates downstream signals which control cell survival, proliferation, growth and other cellular processes. Akt are involved in the phosphorylation of several targets and thus, inactivating their functions. The tuberous sclerosis complex is a heterodimerous of TSC2 (tuburin) and TSC1 (harmatin) and has emerged as crucial integrators of growth factors, nutrients and stress signal such as hypoxia and high low levels of ATP. TSC1 is required to stabilized TSC2 and TSC2, which has the GAP (GTPase-activating protein) activity in its C-terminal domain, integrates different signal form various kinases leading to mTORC1 regulation. In low energy status and in the absence of growth factors the GAP activity promotes the Ras homolog enriched in the brain (Rheb) GTPase activity forming Rheb-GDP and shutting down mTORC1 activity. Rheb In the presence of growth factor Akt is activated and phosphorylates TSC2 in five residues (S939, S981, S1130, S1132 and T1462) resulting in its dissociation form the lysosome where Rheb is localized and thus promoting Rheb activation. In addition, Akt phosphorylates and inhibits PRAS40 leading to mTORC1 activation. In parallel, the activated RTKs stimulate the Ras/ERK/RSK1 signalling axis leading to the inhibition of Rheb GAP activity (Georgescu, 2010), (Kwiatkowski, 2009), (Bartolome & Guillén, 2014). In addition, low cellular energy status (high AMP/ATP ratio) activates the AMPK sensor which phosphorylates different residues of TSC2 increase GAP activity toward Rheb turning down mTORC1 signalling. AMPK also phosphorylate Raptor in a TSC2 independent manner leading to the mTORC1 activation. There is a third mechanisms by which AMPK phosphorylate directly ULK1, which binds and inhibit raptor. There are other cellular stressors such as hypoxia, radiation, high salt concentration, DNA topoisomerase inhibitors and 67 histone deacetylase inhibitors. One of the most important is hypoxia which inhibits mTORC1 by upregulating DNA damage response 1 (REDD1) which inhibits mTORC1 through TSC1/TSC2 (Kim & Guan, 2015), (Paquette et al., 2018). One of the best mTORC1 inductor is amino acids trough the activation of a family of Ras-like small GTPases forming heterodimers (RagA/B/C/D). RagA or RagB forms a heterodimer with Rag C or the RagD and are recruited to the lysosome by the Ragulator. In the presence of amino acids these heterodimers are active and recruit mTORC1 to the surface of the lysosome where GTP- bounded Rheb is located and thus, leading to mTORC1 activation. The Ragulator complex also requires the presence of the vacuolar H+-adenosine triphosphatase ATPase (v-ATPase) complex to sense the amino acids. In the absence of amino acids this complex interacts with Ragulator and when amino acids are present it dissociates. It has been demonstrated that trough the pi3k/akt axis there is also a recruitment of TSC1/2 to the lysosome suggesting that the lysosome is the signalling core for the mTORC1 activation (Kim & Guan, 2015), (Paquette et al., 2018). Growth factors, hormones, insulin, cytokines signalling through RTKs mediates TSC2 phosphorylation (S939 and T1462), which are critical for their spatial location and thus, to its function. These phosphorylation events promote its inhibition by Akt promoting its dissociation from the lysosome and the GTP- bound Rheb accumulation which binds and activate mTORC1 in the lysosome membrane. however in starvation TSC2 is relocated in the lysosome membrane close to mTORC1 (Paquette et al., 2018). In the absence of TSC1 and TSC2 there is a constitutively mTORC1 activation due to the high levels of GTP-Rheb which are involved in cell growth, increasing cell size and protein synthesis. The activity of TSC1/TSC2, Rheb and Raptor are critical and essential for the activation of mTORC1 in presence of growth factor. It has been found that the catalytic domain of MTORC1 only phosphorylates substrates when bound to Raptor (Paquette et al., 2018). 68 In short, mTORC1 activity is influenced by the ATP and amino acids levels, phosphatidic acid and hypoxia. It inactivates the tuberous sclerosis complex protein 2 (TSC2), proline-rich Akt substrate of 40kDa (PRAS40) and the small GTPase Ras homolog enriched in the brain (Rheb) leading to mTOR1 activation and thus, increase of protein synthesis and cell growth. Figure 14|mTOR signalling pathway. There are two mTOR signalling complexes, mTORC1 and mTORC2. mTORC1 is consider a critical sensor and driver of cellular metabolism which respond to nutrients, growth factors and 69 stress signal such as hypoxia and ATP levels. It promotes anabolic process such as protein and lipid synthesis and inhibits catabolism pathways such as autophagy and lysosomal biogenesis. mTORC2 complex is activated by the axis growth factors/RTK/PI3K and regulates cytoskeletal organization, metabolism and cell survival by phosphorylating several kinases. It has been found the relevant function of mTORC2 in the fully activation of Akt in presence of growth factors by the phosphorylation of the S473 residue. DEPTOR, DEP domain containing mTOR-interacting protein; MAPKAP1, also mSIN1, MAPK associated protein 1; mLST8, mTOR associated protein, LST8 homolog; PTEN, phosphatase and tensin homolog; PRAS40, proline rich AKT1 substrate; PDK1, pyruvate dehydrogenase kinase 1; RICTOR, RAPTOR independent complex of mTOR complex 2; TEL2, telomere maintenance 2; TTl1, tubulin tyrosine ligase; TSC, tuberous sclerosis complex; mTORC, mechanistic target of rapamycin complex; TFEB, transcription factor EB; ULK1, Unc-51 like autophagy activating kinase 1; S6K, the ribosomal S6 kinase; 4EBP1, eukaryotic translation initiation factor 4E binding protein; Rheb, Ras homolog enriched in the brain; AMPK, AMP-activated protein kinase; REDD1, DNA damage response 1; ERK, extracellular signal-regulated kinase; TSC, tuberous sclerosis complex; Rictor, rapamycin-insensitive companion of mammalian target of rapamycin; Protor, protiens observer with rictor; HIF, hypoxia-inducible factors; PPARγ, peroxisome proliferator-activated receptor gamma, PGC-1α, peroxisome proliferator-activated receptor gamma coactivator 1-alpha. Downstream signalling Once mTORC1 is activated there is a series of downstream signalling which involved in cell growth, translation initiation and autophagy inhibition. The unc- 51 autophagy activating kinase 1 (ULK1) is supressed by mTOR as well as the Transcription factor EB (TFEB). It is transcription factor which stimulates genes involved in lysosome biogenesis and autophagy-related genes. Its overexpression enhances de lysosomal clearance improving autophagy flux as well as lysosomal exocytosis (Seranova et al., 2017). The eukaryotic translation initiation factor 4E binding protein (4EBP1) is also inhibited, however the 40S ribosomal S6 kinase 1 (S6K1) is activated, increasing protein synthesis. The 70 phosphorylation of 4EBP promotes its dissociation from eIF4E leading to the enhancement of mRNA translation, protein synthesis and cell growth. Moreover, it has been found a link between mitochondria number and function and MTORC1 activation. It activity promotes the expression of PGC1α and YYO which are involve in the regulation of mitochondrial biosynthesis and oxidative function. One of the best studied mTOR function is the inhibition of autophagy through the phosphorylation and inhibition of ULK1, atg13 and FIP200 which form a complex necessary to start the autophagy process. In addition, there is an enhancement of key transcription factors such as hypoxia inducible factor (HIF-1), peroxisome proliferator activated receptor gamma (PPARγ) and PPARγ cofactor 1 alpha (PGC-1α) (Cuyàs et al., 2014), (Ainslie et al., 2016). Little is known about the mTORC2 complex, it has been found that it is insensitive to amino acids nutrients but react in the presence of growth factor by an unkonwn mechanism that involves PI3K and ribosomes. The role of this complex, once it is phosphorylate in Ser2481, is to regulate cell survival, cytoeskeletal organization and metabolism (Cuyàs et al., 2014). 71 6. Autophagy Autophagy is an essential intracellular process, known as self-digestion, involved in survival, differentiation, development and cellular homeostasis. In mammalian cells there are three types of autophagy which differ on the physiological function and the way of cargo formation and delivery to the lysosomes: microautophagy, chaperones-mediated autophagy (CMA) and macroautophagy. The most extended form and studied in this project is macroautophagy. This catabolic mechanism supplies nutrients and energy to the cell to do its vital functions and gets ride of non-needed cellular components such as proteins and lipids, misfolded proteins and damage organelles. It pathway occurs constitutively at low levels for the clearance, but can be induced under physiological stress condition (intra and extracellular stresses) such as energy and nutrient starvation, absence of growth factors, hypoxia, infection and ER stress. Cells degrade their unwanted cytoplasmic material and recycled their monomers which go to biosynthetic process or to produce energy leading to cell survival (Levine & Kroemer, 2008). Autophagy dysfunction may underline mechanisms for several diseases such as neurodegenerative, muscle, cancer and infectious diseases. Autophagy is a multistep process well-regulated by the autophagy-related proteins (ATGs), which includes 1) initiation, 2) membrane nucleation and formation of the phagophore, 3) membrane expansion, 4) autophagosome formation, 5) fusion with the lysosome and 6) degradation of autophagic cargo. (Hansen, Rubinsztein, & Walker, 2018). The initial step is the recruitment of ATG proteins to a subcellular location known as phagophore assembly site (PAS) where takes place the formation of the phagophore, a growing double membrane (vesicle nucleation), around a portion of cytoplasm; then it is extended (vesicle elongation) and engulfs the cargo with the finally fusion of both ends (vesicle completion) forming an double-membrane autophagosome. Several studies suggest that phagophores are nucleated at the PAS on ER membrane named as omegasomes. It has also been found that apart from ER, mitochondria, Golgi, plasma membrane or recycled endosomes are implicated as PAS. The origin of PAS may be cell and context dependent (Dikic & Elazar, 72 2018). Cargo can be proteins aggregation, lipids vesicles, damage organelles etc. This autophagosome needs to fuse with an acidic vacuole which contains the enzymes involved in the cargo degradation (hydrolases) known as lysosome which result in the autolysosome formation. It results from the fusion of the outer membrane of the autophagosome with lysosomes. In the presence of an acidic environment the lysosomes hydrolases degrade the autophagosome inner membrane and the autophagic cargo. Finally, the component parts of the degraded cargo are transported back to the cytosol through lysosomes permeases and then, the cell recycles and re-uses the monomers to form new ones or catabolize to produce energy (Figure 15). It has been identified 32 genes in mammals involved in the autophagic process the so called ATGs and are grouped into six groups: 1) the Unc-51 kinase 1 (ULK1) initiation complex (ULK1-Atg13-FIP2PP-Atg101) for autophagy induction once mTORC1 is inhibited, 2) the class III PI3K nucleation complex (Vsp34-Beclin1-Vsp15-Atg14) for vesicle nucleation, 3) Atg9 for delivery membrane material for the phagophore, 4) the phosphatildylinositol 3- phosphate (PI3P)-binding complex which control the distribution of the Atg12 conjugation system (Atg12-Atg5-Atg16L) and LC3/GABARAP. The Atg12 conjugation system is involved in the conjugation of LC3 where it is cleavaged by the ATG4 protease to form LC3-I and then conjugated with phophatidylethanolamine (PE) to finally form LC3-II. LC3-II is incorporated in the pre and autophagosomal membrane to interact with cargo and be engulfed by the membrane (Pyo, Nah, & Jung, 2012), (Dikic & Elazar, 2018), (Hansen et al., 2018). 79 80 AIMS 81 AIMS Tay-Sachs and Sandhoff are genetic disorders caused by mutations in the α or β (respectively) subunit which conformed lysosomal enzyme β-Hexosaminidase A. However, the molecular pathogenesis of both diseases remains poorly understood due to the low financing available for the study of these diseases. The reduce percentage of affected people worldwide and the difficulty to conduct clinical trials make the financing entities not interested in investing their resources in a monetary unprofitable project. This thesis arises for the need to elucidate the molecular mechanisms that underline these pathologies and the translation of the findings into a treatment. Working with fibroblasts from affected patients derived from skin biopsies our aims were:  To characterize the physiopathology of the patients fibroblasts with mutations affecting the β-Hexosaminidase A enzyme  To evaluated the state of autophagy flux and mTOR pathway in cells from Tay-Sachs and Sandhoff patients  To analyse the mitochondrial morphology, network and function  To generate an HexA knockout (KO) using the CRISPR-Cas9 gene editing system to determine if the pathophysiology of these diseases are associated with the mutations affecting HexA enzyme  To find a treatment with the aim to stop the progression of these pathologies. The challenge is to find a molecule which is currently approved and sold in pharmacies in order to not to conduct clinical trials which would take lots of years to be ready to use 82 83 MATERIALS AND METHODS 84 85 Material and methods Reagents BD Biosciences (New Jersey, USA): anti-cytochrome c BioLabs (New England): BbsI restriction enzyme, Smart cutter Bio-Rad Laboratories Inc. (Hercules, CA, USA): Acrylamide/bisacrylamide solution 37.5:1, Running buffer (TGS), Turbo transfer buffer (TG), Tween-20, Bradford reagent, nitrocellulose membranes 0.2μm Boehringer Mannheim (Indianapolis, Ind): cocktail of protease inhibitors (complete cocktail) Calbiochem-Merk Chemicals (Nottingham, United Kingdom): N-acetyl-DL- leucine Cell Signalling Technology (Danvers, MA, USA): anti-microtubule-associated protein 1A/1B-light chain 3 (LC3), anti-p62, anti-lysosomal-associated membrane protein 1 (LAMP-1), anti-CatB, anti-galectin-3, anti-raptor, anti-p- mTOR Eurofins Scientific (Luxemburgo): gRNA for CRISPR/Cas9 assay Gibco (Invitrogen, Eugene, OR, USA): high glucose media DMEM (Dulbeccoo`s modified media), fetal bovine serum (FBS). Invitrogen/Molecular Probes (Eugen, OR, USA): MitoSOXTM Red, MitoTrackerTM Red CMXRos, Hoechst 3342, 4′,6-diamidino-2-phenylindole (DAPI), Hank's Balanced Salt Solution (HBBS), ATP determination kit Licor (Madrid, Spain) : WesternSure Chemiluminescent substrate Merk Millipore (Burlington, MA, USA): anti-puromycin Novus Biologicals (Colorado, USA): anti-TFEB, anti-Ass1 Promega (USA): FuGene6 Quiagen (Hilden, Germany): Quiagen RNeasy kit, QIAquick gel extraction kit, QIAprep spin miniprep kit Santa Cruz Biotechnology (Santa Cru, CA, USA): anti-GADPH, anti-pDRP1, anti-pAKT, anti-MAPLC3β, anti-catalase, anti-SOD, anti-OGG1/2, anti-HexA Seahorse Bioscience Inc. (Billerica, MA, USA): Mito-stress test kit including oligomycin, FCCP, rotenone & antimycin A, as well as the XFe24 cell culture plates, sensor cartridges and XF base medium. 86 Sigma-Aldrich (St. Louis, Missouri, USA): SDS, TEMED, pounceau reagent, glycerol, paraformaldehyde, CCCP (carbonyl cyanide mchlorophenylhydrazone), Bafilomycin A, Phosphate-Buffered Saline (PBS), DMSO, trypsin, penicillin/streptomycin solution, saponin, red ponceau, ammonium persulfate (APS), L-arginine (A5006), L-leucine (L8000), Lysosome isolation kit ThermoFisher Scientific (Waltham, MA, USA): TMRM, BODIPY 581/591 Vector Laboratories (Burling-game, CA, USA): Vectashield mounting medium 87 Ethical statements Approval of the ethics committee of the University of Seville was obtained, according to the principles of the Declaration of Helsinki and the International Conferences on Harmonization and Good Clinical Practice Guidelines. All the participants in the study provided written informed consent before initiating it. 1. Cell culture After approval of the local ethics committee and informed consent forms form the patients were obtained, patient fibroblasts with Tay-Sachs and Sandhoff were isolate from skin patient’s biopsies. They were obtained according to the Helsinki Declarations of 1964, as revised in 2001. Skin biopsies were placed in 10ml of transport media (PBS1X and antibiotics) and transported at RT. Individual samples were transferred to a sterile 35mm Petri dish and cut in a 5- 10mm2 piece of tissue. Then, it was dissociated with forceps and scalpel transferring to a new 35mm Petri dish with the dermis upside down. The dish was placed inside the culture hood during 5-10 minutes to let the pieces of tissue dry and attach to the dish surface. After that, the samples were covered with DMEM culture 20% FBS and placed in the incubator 37ºC and 5% of CO2 until the fibroblast were propagated. Fibroblasts were cultured in high glucose media DMEM (Dulbeccoo`s modified media) (Gibco, Invitrogen, Eugene, OR, USA) supplemented with 20% fetal bovine serum (FBS) (Gibco, Invitrogen, Eugene, OR, USA) and 1% antibiotics (Sigma ChemicalCo., St Louis, MO, USA). Cells were incubated at 37ºC in a 5% CO2 atmosphere. We worked with two healthy subjects (CTL1, CTL2) acquired in the American Type Culture Collection (ATCC), seven Tay-Sachs patients (four infantile (Inf1- Inf4) and three juvenile (Juv-1-Juv3)) and two Sandhoff patients (SD1, SD2) from Spain. 88 2. Mouse model Mice tissue: brain, brain stem spinal cord and cerebellum obtained from wildtype, knock-out and heterocigous mice for HEXB gene were given by Timothy Cox`s group form Cambridge University (Liebau et al., 2013). 3. Structural mutation analysis The HexA and HexB gene sequences were downloaded from GenBank (Gene ID: 3073 and 3074). The patient mutations were analysed with the web application Mutalyzed (8). To analyse the involvement of the mutations in protein folding, the crystalized 3D structures were obtained from PDB database (HEXA:2GJX and HEXB:1NOU), and they were viewed using PyMOL. To predict the stability of the mutated structures were used to different programs with default values, CUPSAT and SDM, which measure torsion angles and free energy to predict changes in the protein stability. 4. Enzymatic activity of HexA HexA activity was determined as described previously (9). Briefly, total hexosaminidase activity was determined using the 4-methylumbelliferyl-2- acetamido-2-deoxyb-D-glucopyranoside (4-MUG) assay, whereas the HexA enzyme activity alone was determined using the 4-MUGS assay. 5. Proliferation assay Fibroblasts were seeded at 40% confluency in 6-well plates. Several pictures of different 10X-fields were taken using a brightfield microscope and then counted by using with ImageJ Software before and after treatments in order to analysed cell growth. 95 promoter, Cas9, ampicillin (bacterial selection), EGFp (non-bacterial section) and BbsI enzyme restriction cut point. At first, vectors need to be linearized by using the restriction enzyme BbsI. 2ug of vector, 1ul of BbsI (BioLabs) and diluted buffer (Smart Cutter, BioLabs) for 1 hour in a 37ºC-water bath is essential to make an open vector. There is a possibility to both endings join again. Therefore, a nucleotide adding reaction needs to be done. We used 10ul of open-vector, 1 μl of Phosphatase enzyme (), 2ul pf Phosphatase Buffer and 7ul of H20d, and leave it 30 minutes in a 37ºC water bath. Then, we needed to purify the digested vector running a 0,7% agarose gel electrophoresis for 45 minutes at 95 voltage. After checking the band using UV light, it is cut and the plasmid is isolated using a QIAquick Gel Extraction Kit (Qiagen). Finally, the dna is eluted from the columns using H20d. The next step is the annealing of each pair of oligos. 1ul of each oligo 100mM (forward and reverse) were added to 1ul of T4 Ligation Buffer NEB, 0,5 μl of T4 NEB and 6,5 μl H2Od. The annealing is made by using the thermocycler with the following parameters: 37ºC for 30 minutes, 95ºC for 5 min and then ramped down to 25ºC at 5ºC/minute. After that, the set up ligation reaction is carried out by adding into a tube 50ng of open vector, 200ng of oligos annealed, 2ul Ligation Buffer and 1ul of Ligation enzyme up to a final volume of 20ul. The tube is let overnight at 16ºC. The vector is amplificated thought bacterial transformation using NEB 5-alpha competent bacteria. 10ul of plasmid (ligation reaction) and 35 μl of bacteria are adding into a tube into a 42ºC water bath for 45 seconds following of 2 minutes on ice. 300ul of SOC media is added to the tube and keep it for 45 minutes in a 37ºC shaker incubator at 225rpm. 250ul of the tube is spread into an agar plate and keep it overnight at 37ºC. Then, some bacteria colonies are picked up and incubated with LB media and Ampicillin (1:1000) overnight at 37ºC in a shaker incubator. In this step we are doing the selection of the bacteria which have incorporated the plasmid with the ampicillin resistant gene in the vector. Plasmids are isolated form the bacteria using QIAprep Spin Miniprep kit (QUIAGEN) and checked by running an electrophoresis. The vectors are sent to Eurofins for DNA sequencing to ensure that the vector has the insertion and if 96 its orientation is correct. After sequencing, the sequence is checked using Snapgene Software. HeLa cells were seeded in a 6cm dish at low confluency and after 24 hours the cells are transfected using FuGENE6 (). Cells which have the plasmids express GFP, so we used fluorescence activated-cell sorting (FACS) and 1 cell per well is seeded in a 96-well plate. After 1 or 2 weeks cells start to form colonies, they are splited to amplify the colonies. Finally, we check the cells by WB using an anti-Hexa antibody (Figure 18). Figure 18| Steps followed in the CRISPR/Cas9 assay. 97 16. Measurement of mitochondrial generation of superoxide Mitochondria is considered the major intracellular source of ROS, due to during mitochondrial respiration many ROS, such as anion superoxide, are produced as an incidental product. Immunofluorescence analysis was performed using MitoSOXTM Red mitochondrial superoxide indicator (Invitrogen/Molecular Probes, M36008) which is a florigenic dye for the detection of the superoxide in the mitochondria. MitoSOX™ Red reagent is live-cell permeant and is rapidly and selectively targeted to the mitochondria. Once in the mitochondria, MitoSOX™ Red reagent is oxidized by superoxide and exhibits red fluorescence. Fibroblasts were cultured in six-well plates on the top of cover slips and incubated with 5μM MitoSOXTM Red in DMEM 20%FBS, 1% antibiotics for 10 min at 37ºC and 5% CO2. After incubation, cells were washed with PBS1X and fixed with 3,8% PFA for 5 min. Finally, the nucleus was stained with DAPI and the images were analysed using DeltaVision system (Applied Precision; Issaquah, WA, USA) with an Olympus IX-71 microscope. 17. Lipid peroxidation Fibroblasts were cultured on coverslips and incubated with 1 μM C11-Bodipy (BODIPY® 581/591 C11) for 30 min at 37 °C. Coverslips were then rinsed with PBS and mounted onto slides as describe above for analysis with a fluorescence microscope. Fluorescent intensity was measured using the Image J software (National Institutes of Health, Bethesda, Maryland, USA). 18. Antioxidant enzyme activity Enzyme activity of antioxidants was determined in 1x106 cells. Catalase activity was determined in cellular lysates by monitoring H2O2 decomposition at 240nm. Superoxide dismutase activity was determined based on inhibition of the formation of NADH-phenazine methosulfate-nitroblue tetrazolium formazan (Kakkar, Das, & Viswanathan, 1984). 98 19. Lipid hydroperoxides The FOX assay was carried out according to the method previously reported [9]. The FOX reagent was prepared by mixing in the following order: 90 ml methanol, 88 mg BHT, 10 ml 250 mM H2SO4, 9.8 mg ammonium ferrous sulphate hexahydrate and 7.6 mg Xylenol Orange. To 320 l of sample, 680 l of FOX reagent were added and the solution was incubated for 30 min at 37 ◦C with gentle shaking. After a short high-speed centrifugation (3000 × g for 1 min at room temperature), sample absorbance was read at 560 nm against the blank (0.9% NaCl and FOX reagent). For hydroperoxide quantification, a serial standard dilution of hydrogen peroxide was used (Jiang, Hunt, & Wolff, 1992). 20. Measurement of citrate synthase activity The specific activity of citrate synthase in whole-cell extracts prepared from control and patient fibroblasts was measured at 412 nm minus 360 nm (13.6 mM−1 cm−1) using 5,5-dithio-bis(2-nitrobenzoic acid) to detect free sulfhydryl groups in coenzymeA as described previously (Bullon et al., 2011). 21. Analysis of mitochondrial network and morphology Fibroblasts were incubated with 10nM of Mitotracker red CMXRos (Invitrogen, M7512) for 20 minutes at 37ºC. After washing with PBS1X the culture media was changed for 30min and then cells were fixed in 4% PFA for 15 min at RT and the nuclei was stained with DAPI (1μg/mL). Samples are analysed using MICROSCOPIO. MEDICCION DE FLUORESCENCIA (TESIS) In addition, a standard immunofluorescence was performed using an antibody against TOM-20 (mitochondrial membrane marker). 22. Mitophagy analysis Fibroblast were seeded in coverslips in 6-well plate, fixed in 3,8% PFA for 5min at RT and permeabilized with 0,5% saponin for 5 min. Mitophagy analysis were 99 performed by standard immunofluorescence techniques by using LC3B (autophagosome marker) and cytochrome c (mitochondria marker). Colocalized signal of LC3B/cytochrome c was assessed by using DeltaVision system (Applied Precision; Issaquah, WA, USA) with an Olympus IX-71 microscope. 23. TMRM assay Tetramethylrhodamine methyl ester (TMRM, Life Technologies) was used to determine the mitochondrial membrane potential. Cells were grown in 6cm glass bottom image dish, washed with PBS and then incubated in Imaging Buffer (HBSS 1X, Glucose 5mM, HEPES 10mM, MgCl2 1mM, CaCl2 1.26mM and H2Od) with 25nm TMRM for 25 minutes at 37ºC. Images were taken using DeltaVision system (Applied Precision; Issaquah, WA, USA) with an Olympus IX-71 microscope. After that, 1uM of CCCP were added and data were obtained after 1 min. Images obtained were analysed using ImageJ Software (National Institutes of Health, Maryland, USA). 24. Analysis of mitochondrial respiratory function using Seahorse extracellular flux analyser Mitochondrial respiratory function and profile of controls and Tay-Sachs and Sandhoff fibroblasts were measured by using Mito-stress test assay by XFe24 extracellular flux analyser (Seahorse Bioscience, Billerica, MA, USA). The seeding density was 30.000 cells/well and the compounds concentration were 1 μM oligomycin, 2 μM FCCP and 1 μM and 2.5 μM rotenone & antimycin A, respectively. Fibroblasts were seeded in XFe24 cell culture plates in 100 μL growth medium and placed in 37°C incubator with 5% CO2. After cells adhered within 1 h, 150 μL growth medium were added and cells were returned to 37°C incubator with 5% CO2. After 24 h incubation, growth medium from each well was removed, leaving 50 μL of media. Cells were washed twice with 500 μL of prewarmed assay medium (XF base medium supplemented with 10 mM glucose, 1 mM glutamine and 1 100 mM sodium pyruvate; pH 7.4). Then, 450 μL of assay medium (500 μL final) were added. Cells were incubated in 37°C incubator without CO2 for 1 h to allow preequilibration with the assay medium. Pre-warmed oligomycin, FCCP and rotenone & antimycin A were loaded into the injector ports A, B and C of sensor cartridge, respectively. The final concentrations of injections were as follows: 1 μM oligomycin, 2 μM FCCP and 1 μM rotenone & 2.5 μM antimycin A. The cartridge was calibrated by the XFe24 analyzer and the assay was carried out using cell Mito-stress test assay protocol as described by Nicholls et al.574. When the experiment was finished cells number of each well were calculated using the Neubauer chamber. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured followed by the sequential addition of oligomycin, FCCP and rotenone & antimycin A. This approach allows determining different parameters of mitochondrial function such as basal respiration, maximal respiration, spare respiratory capacity and ATP production. Basal respiration: The cell capacity of utilization the mitochondrial respiration under baseline conditions. It is calculated as the value of the last rate measurement before oligomycin injection minus the non-mitochondrial respiration rate. Maximal respiration: The maximum rate of mitochondrial respiration that the cells can achieve. It is calculated as the value of the maximum rate measurement after FCCP injection minus the non-mitochondrial respiration rate. Spare respiratory capacity: The cell capability to respond to an energetic demand. It is an indicator of cell fitness or flexibility. It is calculated as the value of the maximal respiration minus the basal respiration. ATP production: Measure the ATP production by the mitochondria. It is calculated as the value of the last rate measurement before oligomycin injection minus the minimum rate measurement after oligomycin injection. Data analysis of Seahorse assays 101 The XF Mito-stress test report generator automatically calculates the XF Mitostress test parameters from Wave data that have been exported to Excel. Respiration and acidification rates are presented as the mean ± SD of 3 independent experiments in all experiments performed with 3 replicate wells in the Seahorse XFe24 analyzer. 25. ATP levels ATP levels were determined by a bioluminescence assay using an ATP determination kit from Invitrogen-Molecular Probes (Eugene, OR, USA) according to the manufacturer’s instructions. 26. Microarrays Total RNA from fibroblasts and right-cerebrum of Sandhoff mice (WT, HET and KO of HEXB gene) were extracted using the RNeasy kit (RNeasy, QIAGEN, Valencia, CA) and then the quality of total RNA for array analysis was determined using an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). Biological replicates were synthetized, amplified, purified and the corresponding ss-DNA was labelled with biotin according to the manufacturer’s protocol (Affymetrix). 100 ng of total RNA was hybridized to 5.2 μg of labelled cDNA. Then, hybridation, rinsing and scanning of the signal (Scanner 3000 7G, Affymetrix) were performed. Array data were processed using Affymetrix® Genechip® Command Console® 2.0 with respect to background subtraction and normalization. We used the Affymetrix Microarrays ClariomTM D Arrays Human and Mouse, which includes 65956 genes. The raw data were analysed by the Affymetrix software Trancriptome Analysis Console (TAC) which analyse and visualize global expression patterns of genes, exons, pathways, and alternative splicing events. Expression values were normalized by SST (Signal Space Transformation)- RMA (Robust Microarray Analysis; Irizarry et al. 2003). Many comparisons of transcription levels were made between the different conditions in fibroblasts 102 and mice brain tissues, as are schematized in the Figure 19. Three replicates of each condition were made for every analysis. Figure 19| Comparison of transcription levels between Tay-Sachs and Sandhoff fibroblasts compared with healthy ones and HEXB KO mice compared with HET and WT ones. Then, a fold change and p-value was calculated for every gene by an unpaired test one-way (single factor) using the NMATH package. A gene was considered as differentially expressed when it had a fold change equal or higher than 1.5, and a p-value equal or lower than 0.05. Then, a functional enrichment was performed with the differentially expressed genes using the functional annotation tool from the DAVID web site (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3381967/). We found a number of annotation categories. To analyse the expression profile for the genes included in these categories, a heatmap was created for every annotation category, using the heatmap.2 function in the gplots R package, and considering normalized values by z-scores, and making a clustering based on the expression profiles and the average method. Finally, a mean of the expression values in every annotation category was calculated and it was again plotted in a heatmap (Figure 20). 103 Figure 20| Schematic representation of the Microarrays assay. 27. Microscopy -Widefield microscopy was performed using: Leica DMi1 brightfield microscope (Leica Microsystems GmbH, Wetzlar, Germany) -Fluorescence microscopy was performed using: DeltaVision system (Applied Precision; Issaquah, WA, USA) with an Olympus IX-71 microscope -Electron microscopy was performed using: Zeiss LEO 906 E (Zeiss, Oberkochen, Germany) transmission electron microscope. 104 28. Statistical analysis Data in the figures is given as mean ± SD. Data between different groups were analysed statistically by using ANOVA on Ranks with Sigma Plot and Sigma Stat statistical software (SPSS for Windows, 19, 2010, SPSS Inc. Chicago, IL, USA). For cell-culture studies, Student’s t test was used for data analyses. A value of p<0.05 was considered significant. 111 Patient Maternal Mutation Paternal Mutation Inf1 c.409C>T Point mutation Premature stop codon Protein stability and active site are affected c.1073+1G>A Point mutation Splicing site Inf2 IVS4+5G>A (c.459+5G>A) Point mutation Near splicing site IVS4+5G>A (c.459+5G>A) Point mutation Near splicing site Inf3 c.1274_1277dupTATC Point mutation Premature stop codon Protein stability and active site are affected c.785A>G Point mutation Protein stability and active site are affected Juv1 c.536A>G (p.His179Arg) Point mutation Protein stability and active site are NOT affected Described mutation c.533G>A (p.Arg178His) Point mutation Protein stability and active site are affected Described mutation Juv2 c.(533g>a) (p.Arg178His) Point mutation Protein stability and active site are affected Described mutation c.915-917delCTT Point mutation Juv3 c.1073+1G>A Point mutation Splicing site c.1305C>T p.Y435Y Point mutation Protein stability and active site are NOT affected p.Trp57Cys (c.171G>C) c.1509-26G>A (IVS12-26G>A) Point mutation 112 Table 7| Mutations found in patients. Mutations where there is not specified if they affect the stability and the active site of the enzyme is unknown. Apart from the nine patients, another more infantile patient (Inf4) and two adult patients (Adl1 and Adl2) have been used in several experiments. SD1 Point mutation (propeptide) Protein stability affected Active site NOT affected Splicing site Protein stability and active site are NOT affected SD2 c.1021_1024delAGTG Point mutation Premature stop codon Protein stability and active site are affected IVS12-26G>A Point mutation Splicing site Protein stability and active site are NOT affected Inf4 IVS4+5G>A (c.459+5G>A) Point mutation Splicing site Protein stability and active site are NOT affected IVS4+5G>A (c.459+5G>A) Point mutation Splicing site Protein stability and active site are NOT affected Adl1 p.L484P Point mutation Protein stability affected Active site NOT affected Described mutation Not identified Adl2 p.L484P Point mutation Protein stability affected Active site NOT affected Described mutation Not identified 113 Most of alterations are point mutations which affect the protein stability except form the one in allele 1 of Juv1 and allele 2 in Juv2. The allele 1 of Inf1, Inf3 and SD2 suffer from point mutations which lead to a premature stop codon and thus, the stability and active site of the enzyme are significantly affected. Moreover, there are several point mutation which are place in a splicing site or near them such as Inf2 and Inf4 in both alleles and alleles 1 form Juv3 and allele 2 form Inf1, SD1 and SD2. Additionally, it has been found 4 described mutation belonging to allele 1 from Juv2, both alleles form Juv1 and Adl1 and Adl2 mutations (Table 7). The position of each mutation along the HEXA of HEXB gene is shown in the Figure 20. 114 Figure 20| Location of the point mutation of both alleles of each patient under study along the A) HEXA and B) HEXB gene. Each colour corresponds to an allele of a patient; grey colour is the mutation located in introns. To evaluate the possible effects of the mutations on the protein structure a three-dimensional (3D) structure for the α-subunit and β-subunit was performed. In the Figure 21 are represented the exons mutations of the patients. In image A it is shown a cartoon representation of the α-subunit of HexA enzyme with point mutations. Juv1 point mutations in both alleles (red) affect two amino acids near the active site (green) as well as mutation in allele 2 from Inf3 mutation (light blue). It is also shown in image C in a globular representation of the α-subunit where the mutations can be seen in the protein surface. Changes in Juv2 also occur very near of the active site (dark blue, red). Mutation in allele2 from Juv3 and mutations in Adl1 and 2 affect farther places (pink and yellow, respectively). Point mutations which generate premature stop codons are represented in the image B showing the structure affected by allele1 from 115 Inf1 (yellow) and allele 1 from Inf3 (orange). It is also shown the structure art that are loss due to the mutations (grey). Sandhoff patient mutations are represented in the image D where it is displayed the β-subunit of HexA enzyme. Point mutation in allele1 from SD2 leads to a stop codon affecting much of the protein (orange) which drives to the loss of a part of the sequence (grey). Figure 21| Three-dimensional structure models for HEXA (PDB:2GJX) and HEXB (PDB:1NOU) highlighting the patient mutations. A) HEXA gene point mutations in coding sequences, different colours represent amino acid substitutions. B) HEXA frameshifts coming from two patients (orange and yellow). C) Globular representation of α-subunit showing the protein surface and highlighted critical region for the hydrolysis of GM2 gangliosides (green). D) Mutations of Sandhoff patients (SD1, SD2) in the β-subunit of the HexA enzyme. It is also shown the structure art that are loss due to the mutations (grey). *Stop codons. 116 These alterations in protein structure result on the loss of the stability which is associated to the decreasing in the HexA enzymatic activity in blood cells compared with reference values of healthy controls (Figure 22 A). Due to fibroblast cell culture models provide satisfactory information about the molecular mechanisms underlying the pathological alterations in genetic diseases, we determined the metabolic status of patient fibroblasts. Tay-Sachs fibroblasts showed reduce growth rates (Figure 22 B) and abnormal morphology with the intracellular accumulation (Figure 22 B) compared with healthy fibroblasts. 117 Figure 22| A) Enzymatic activity of HexA in homogenates from control and patients skin fibroblasts. B) Growth curve of Tay-Sachs patient fibroblast compared with healthy ones during 120 hours. It is shown a reduce growth rate in patient cells. C) Morphological changes of patient fibroblasts compared with the control one, where it can be appreciated the cytosolic accumulation. 118 119 2. Autophagic flux is impaired in fibroblast from Tay- Sachs and Sandhoff patients The accumulation of non-degraded material within lysosomes is the most relevant sign of Tay-Sachs and Sandhoff diseases. The cellular machinery which is involved in this self-digestion is autophagy which its correct activity is essential to maintain cellular functions. Patients have mutations in HEXA or HEXB genes which cause the absence or partially activity of the enzyme βhexosaminidase A. Because mutant in both genes can affect the degradative cellular process we firstly evaluated the expression levels of the two major Autophagic marker proteins using Western blotting: Lc3 and p62. The microtubule-associated protein 1A/1B-ligth chain 3 (LC3) is a 17kDa soluble protein is distribute in all mammal cells. LC3-I is the free cytoplasmic form which, by following a conjugation with phosphatidylethanolamine produces the LC3-II form which is recruited to the membrane of autophagosomes. When they are fused with lysosomes to form an autophagolysosomes (also now as autolysosomes) LC3-II is degraded by lysosomal hydrolases along with the cytoplasmic cargo which is tagged with p62 protein (Tanida, Ueno, & Kominami, 2008). Lc3-I is converted to Lc3-II during the formation of the autophagosome and shows the autophagy levels and p62 is a protein which tags the substrates that need to be degraded and shows the degradation level of the autophagosomes. In order to determine the autophagy flux status the expression levels of both proteins needs to be quantified. LC3-II protein is highly increased and there is a high level of the LC3-II/LC3-I ratio which means increase autophagy; which it is supposed to be good in these diseases to get rid of all the unwanted cellular waste. However, fibroblasts from patients show high levels of p62 protein, which means that the autophagosome cargo isn’t degraded and is still with the lysosomes (Figure 22). 120 Figure 22| Autophagy flux is impaired in Tay-Sachs and Sandhoff fibroblasts. Autophagic protein expression levels of p62, LC3-I (top panels, top band) and LC3-II (top panels, bottom band) were determined in fibroblast culture form healthy control subjects (CTL) and (A) Tay-Sachs, infantile (Inf) and Juvenile (Juv) and (B) Sandhoff (SD) patients using Western blot analysis, as described in Materials and Methods section. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Results are expressed as mean ± SD of three separate experiments. Significance of LC3II/LC3I expression levels of patients respect to control fibroblasts is represented as 127 Ultrastructural analysis by electron microscopy of fibroblasts from patient fibroblasts confirmed the presence of a high dense cytoplasm full of doublemembrane vesicle structures (autophagosomes), multilamellar bodies, lipofuscin and non-define structures (Figure 26). This increment in autophagosome number correlates with the increase level so f LC3II in western blot (Figure 22 ) and immunofluorescence assays (Figure 25 ).Lamellar bodies are round or oblong organelles which contain lipids such as phospholipids, glucosylceramides, sphingomyelin and cholesterol. They are considered as lysosomes related organelles because of their enzymatic content and vacuolar H+-ATPase leading an acidic pH (lie Raymond et al., 2008). 128 129 Figure 26| Electron microscopy of control and patient fibroblasts. A) Control fibroblasts showing mitochondria with typical ultrastructure (purple arrow) and several lamellar bodies and patient fibroblasts showing high levels of lamellar bodies (white arrows) and autophagosome accumulation (black arrows). The number of autophagosomes per cell was plotted. Results are 130 expressed as mean ± SD of three separate experiments. Significance of patient cells respect to control cells is represented as***P<0,001.B) Several EM images of infantile Tay-Sachs fibroblasts show huge number of lamellar bodies with good morphology and well-developed (I, II and III) and damage or empty (IV). Some non-defining structures have been found in the cytoplasm of patient fibroblasts (V). [Scale bar= 10μm]. Given that reduction of the fusion between autophagosome and lysosome or inefficient lysosomal degradation are associated to autophagosome accumulation and autophagic flux the autophagosome formation was monitored using the autophagy flux detector PremoTM Autophagy Tandem Sensor. It consists on a construct encoding for the LC3-II protein in tandem with GFP and RFP. Autophagosomes are positive for both fluorescence proteins; however when the fusion with the lysosome takes place the green fluorescence disappear and thus, autolysosomes are only positive for red fluorescence. As we thought, patient fibroblasts show high levels of colocalization of LC3-GFP and RFP which means autophagosome accumulation due to the inability to fuse with lysosomes. Apart from these findings, the most relevant feature found is the increment of the puncta size. Autophagosomes size is very huge by reason of the high intra-lysosomal accumulation of GM2 gangliosides and its failure to fuse with lysosomes (Figure 27). 131 132 Figure 27| Monitoring autophagolysosome formation in Tay-Sachs fibroblasts. Control and Tay-Sachs patient fibroblasts were infected and incubated overnight with Baculovirus expressing LC3-GFP-RFP using Bacman technology. Patient cells show huge autophagosome accumulation with an increment in their size inferred form the high colocalization of GFP and RFP. Autophagosomes are positive for green and red fluorescence (white arrow), whereas autolysosomes and lysosomes are only positive for red fluorescence (red arrow). To quantify the number of autophagosome per cells, colocalized puncta (yellow puncta) were measured with the Red and Green puncta Colocalization ImageJ macro (“Green and Red Puncta Colocalization [ImageJ Documentation Wiki],” n.d.). Data are shown in the plot as the mean ± SD of three independent experiments. *P<0,05;***P<0,001 and ***P<0,0001 between CTL and each patient. [Scale bar = 6μm]. In order to assess the lysosomal biogenesis status we did an immunostaining for the Transcription Factor EB (TEFB). TFEB plays an essential role in the regulation of key cellular processes such as lysosomal biogenesis and autophagy. In healthy cells with a proper autophagic flux TFEB is phosphorylated (inactive) and diffuse in the cytoplasm, however inhibition of lysosomal function result in the TFEB dephosphorylation (active) and translocation from the cytosol to the nucleus where it stimulate the transcription of gene involve in lysosomal biogenesis (Napolitano & Ballabio, 2016). As we expected, there is a high TFEB puncta concentration in the nucleus of patient fibroblasts which indicates high transcription levels of genes involve in lysosomal production (Figure 28). Defective autophagy in patient cells leads to an accumulation of autophagosomes with non-degraded material inside the cell cytoplasm. Due to the inability for discard these unwanted products, cells enhance the autophagy flux by stimulating genes implicated in the lysosomal biogenesis to solve its defects. However, the enhancement of autophagy in these diseases is not the key to fix the deficiency because due to the lack (or partially lack) of the β-Hexosaminidase A enzyme activity the accumulated GM2 gangliosides will continue being aggregated. 133 134 Figure 28| Lysosomal biogenesis is up-regulated in Tay-Sachs and Sandhoff fibroblasts. There is an increase in nuclear TFEB localization in patients fibroblasts compared with control ones, which means that TFEB is stimulating the transcription of gene involve in lysosomal biogenesis in mutant fibroblasts. Data are shown in the plot as the mean ± SD of three independent experiments. **P<0,01 and ***P<0,0001 between CTL and each patient. [Scale bar =15μm]. 135 136 3. Lysosomal permeabilization is associated with Tay-Sachs and Sandhoff diseases Degradation of autophagic substrates takes place inside the autolysosome once the autophagosome fuse with the lysosome in the last autophagic step. High impaired autophagic flux along with high autophagosome accumulation can be a consequence for the decrease level of autophagosome-lysosome fusion or inefficient lysosomal substrates degradation (Mizushima, Levine, Cuervo, & Klionsky, 2008) . Consequently, we hypothesized the existence of lysosomal permeabilization events in patient cells which makes the degradation step more challenging. The degradation is carried out by acidic proteases such as cathepsins; these hydrolases are a cysteine family including many enzymes such as cathepsin B, C, D, H and L. We first analysed the Catepsin B (CatB) expression because in a recent research about another LSD called as Papillon Lefevre it has been found an increase level of CatB expression due to the lack of another lysosomes protease (Catepsin C) (Bullón et al., 2018). According to this, we determined if the impaired autophagic flux in patient cells was secondarily associated with further lysosomal dysfunction. Interestingly, active CatB expression levels were increased in Tay-Sachs and Sandhoff fibroblasts (Figure 28) as well as in Sandhoff mouse model tissues (Figure 29). In Tay-Sachs patients we have associated this increment with the reduction of the mature HexA form. 143 Figure 31| Immunostaining of HexA in control and Tay-Sachs (A) and Sandhoff (B) fibroblasts. The patient cells images show decrease levels of HexA with low colocalization with LAMP-I, which implies the lysosomal 144 permeabilization. Results are expressed as mean ± SD of three separate experiments. *P<0,05 and **P< 0,01 between patients and control cells. In addition, in order to reinforce our findings about the leaky lysosomes we did an immunostaining against galectin-3 to detect whether the galectin puncta colocalize with the autophagosome/lysosome membrane (LAMP-I) (Figure 32). Galectins are soluble carbohydrates-binding lectins which easily bind βgalactoside sugars located through the cytosol, nucleus and in the extracellular space. Galectin-3 has been used as a method to detect lysosome membrane permeabilization because they are recruits from the cytosol to the sites of lysosome leaky, targeting damage lysosomes. It is demonstrated that the luminal tails of LAMP-I and LAMP-II form an N-acetylgalactosamine-rich carbohydrate shield to which galectin-3 binds with its carbohydrate recognition domains (Aits et al., 2015). As we expected, in patient fibroblasts there were high level of galectin-3 colocalized with LAMP-II, which means vacuole lysis and/or permeabilization. [Scale bar=15μm]. 145 146 Figure 32|Immunostaining of control and Tay-Sachs and Sandhoff cells with galectin-3. Representative fluorescent imagen of fibroblasts from control subjects and patients staining with an anti-galectin-3 and LAMP-I antibody. The images show colocalization of galectin-3 (green) and LAMP-I (red) in pathologic cells which means the presence of leaky lysosomes. Results are expressed as mean ± SD of three separate experiments. *P<0,05; **P< 0,01;***P<0,001 and ****P<0,0001 between patients and control cells. [Scale bar=15μm]. In agreement, cellular fractionation with isolation of lysosomes indicated an increase in the cytosolic levels of HexA and a concomitant reduction in the lysosomes fraction in fibroblasts from Tay-Sachs patients compared with control subjects with also increased of CatB in the cytosol fraction (Figure 33). 147 Figure 33|CatB and HexA level in the cytosolic and lysosome fraction control subjects and Tay-Sachs cells. Fractionation was performed as described in Materials and Methods section and cellular lysate were processed to isolate the cytosolic and lysosome fractions. Translocation of CatB and HexA to the lysosomal to the cytosol was analysed by Western blotting. The graph show the increase CatB and HexA levels in the cytosolic fraction when compared with controls. Results are expressed as mean ± SD of three separate experiments. **P< 0,01 and ***P<0,001 between patients and control cells for HexA expression. aP<0,05; aaP<0,01 and aaaP<0,001 between patients and control for CatB. 148 149 4. mTOR pathway is downregulated The mammalian target of rapamycin (mTOR) regulates metabolism, cellular growth, protein synthesis, tissue repair, cytoskeletal organization, cellular survival, autophagy and aging. To explore the mTOR signalling pathway we studied the status of mTOR because it is a well-known regulator of autophagy and as we shown in the first part of the Results section, the autophagy flux in Tay-Sachs and Sandhoff fibroblast is impaired with high accumulation of autophagosomes. As we expected, a downregulation of pmTOR and pAkt were observed in Tay-Sachs cells (Figure 34 A) and Sandhoff cells (Figure 34 B) compared with control cells. 150 Figure 33| mTOR pathway is downregulated in Tay-Sachs and Sandhoff cells. pmTOR and pAkt expression levels were determined in fibroblasts from Tay-Sachs (A) and Sandhoff (B) using western blot analysis as describe in Methods section. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Data represent the mean ± SD of three independent experiments. **P< 0,01;***P<0,001 and ****P<0,0001 between patients and control cells. In addition, pmTOR expression levels were downregulated in Sandhoff mouse model (KO) compared with WT and HET mice (Figure 34). Figure 34| mTOR pathway is downregulated in Sandhoff mouse model. pmTOR and pAkt expression levels were also determined in with brain, brain 151 stem, spinal cord and cerebellum tissue from Sandhoff mouse model (KO), HET and WT mice using western blot analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Results are expressed as mean ± SD of three separate experiments. *P<0,05 and**P< 0,01 and ***P<0,001 between WT and KO and HET mice. WT (Wild-type):+/+; KO (Knock-out): -/-; HET (heterozygous):+/-. The mTORC1 is highly regulated and it is found that the activity of TSC1/TSC2, Rheb and Raptor proteins are critical and essential for its activation. Between them, Raptor deserves special mention due to its binding is needed for the activation and thus for the mtorc1 to phosphorylates its downstream substrates. (Paquette et al., 2018). For this reason we analysed the protein expression levels of Raptor by western blot and, as we expected raptor was downregulated in Tay-Sachs and Sandhoff fibroblast compared with control cells. In addition, we checked its expression level in the brain tissue of Sandhoff mouse model compared with WT mice and it was also downregulated (Figure 35). In conclusion, the PI3K/AKT axis is downregulated; in the absence of activated Akt TSC1/TSC2 complex is located in the lysosome membrane where they are promoting by its GAP activity the formation of Rheb-GDP and shutting down mTORC1 activity. Furthermore, another way to confirm the mtorc1 downregulation is by the low Raptor protein expression which is essential in the activation of mtorc1. (Kim & Guan, 2015). 152 Figure 35| Raptor protein is downregulated in patient fibroblasts and Sandhoff mouse model. Raptor protein expression levels were determined in fibroblast from Tay-Sachs (A) and Sandhoff and in brain tissue of Sandhoff mouse model (B) using western blot analysis as descried in Methods section. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Data represent the mean ± SD of three independent experiments. *P< 0,05 and ****P<0,0001 between patients and control cells. In mice, *P<0,05 and **P<0,01 and aP<0,05 between KO and HET with the first WT and the second 255 APPENDIX 256 257 Appendix 1. Publications 1. Fabiola Marín-Aguilar*, Beatriz Castejón-Vega*, Elísabet Alcocer-Gómez, Matthew A. Cooper, Eloísa Andújar-Pulido, Mónica Pérez-Alegre, Jordi Muntané, Antonio J. Pérez-Pulido, Avril A. B. Robertson, Jesús Ruiz- Cabello, Pedro Bullón, Mario D. Cordero “NLRP3 inflammasome inhibition by MCC950 in aged mice improves health via enhanced autophagy and PPARα activity”. Journal of Gerontology: Biological Sciences, 2019 *Equal Contribution. 2. Fabiola Marín-Aguilar, Ana V. Lechuga-Vieco, Diego Cañadas-Lozano, Elísabet Alcocer-Gómez, Beatriz Castejón-Vega, Javier Lucas, Carlos Garrido, Alejandro Peralta-Garcia, Antonio J. Pérez-Pulido, Alfonso Varela- López, José L. Quiles, Bernhard Ryffel, Ignacio Flores, Pedro Bullón, Jesús Ruiz-Cabello, Mario D. Cordero “NLRP3 inflammasome suppression improves longevity and prevents cardiac aging in male mice”. Aging Cell, 2019 3. Diego Cañadas-Lozano, Fabiola Marín-Aguilar, Beatriz Castejón-Vega, Bernhard Ryffel, José M. Navarro-Pando, Jesús Ruiz-Cabello, Elísabet Alcocer-Gómez, Pedro Bullón, Mario D. Cordero. “Blockade of the NLRP3 inflammasome improves health and lifespan in obese mice”. Geroscience 2019 4. Bullón P*, Castejón-Vega B*, Román-Malo L, Jimenez-Guerrero MP, Cotán D, Forbes-Hernandez TY, Varela-López A, Pérez-Pulido AJ, Giampieri F, Quiles JL, Battino M, Sánchez-Alcázar JA, Cordero MD. “Autophagic dysfunction in patients with Papillon-Lefèvre syndrome is restored by recombinant cathepsin C treatment”. Journal of Allergy and Clinical Immunology, 2018 Feb 2. pii: S0091-6749(18)30144-1. *Equal Contribution 258 5. Alcocer-Gómez E, Castejón-Vega B, Cordero MD. “Stress-Induced NLRP3 Inflammasome in Human Diseases”. Adv Protein Chem Struct Biol. 2017;108:127-162. 259 2. Mettings 1. Oral Presentation: 1. “Tay-Sachs and Sandhoff diseases”. III Jornadas de Experiencias Compartidas. University of Seville, Spain, October, 2019. 2. “Autophagy dysfunction and Lysosomal permeabilization in Tay- Sachs and Sandhoff Diseases”. European Network of Multidisciplinary Research and Translation of Autophagy knowledge Annual meeting of Transautophagy. Sofia, Bulgary. April, 2019. 2. Poster Presentation: 1. “Tay-Sachs and Sandhoff: two rare diseases with mitochondrial dysfunctions”. IX Congreso Internacional de Medicamentos Huérfanos y Enfermedades Raras, Seville. February, 2019 2. “Mitochondrial dysfunctions in Tay-Sachs and Sandhoff diseases”. VI Meeting on Genomic Medicine and Oncology of Genyo. Genyo Center, University of Granada, Spain. December, 2018. 3. “Autophagic dysfunction in patients with Papillon-Lefevre syndrome is restored by recombinant cathepsin C treatment”. 1st Meeting on Vesicular Biology. University of Coimbra, Portugal. February, 2018. 4. Autophagic dysfunction in Papillon Lefèvre”. V Meeting on Genomic Medicine and Oncology of Genyo. Genyo Center, University of Granada, Spain. December, 2017. 260 261 AGRADECIMIENTOS 262 263 Agradecimientos Siempre tuve claro dedicarme a la ciencia, pero nunca pensé que llegaría este momento, el de ser Doctora. Me dieron la oportunidad de realizar el doctorado para el estudio de estas enfermedades, el cual supuso un reto en el que puse todo mi interés y tiempo. No puedo catalogar este recorrido como algo fácil, pero si puedo afirmar que durante todo este tiempo he disfrutado de cada momento y crecido como persona y como científica. Este trabajo no hubiese sido posible sin la participación de personas e instituciones, los cuales siempre me han facilitado las cosas. Por ello, es para mí un placer agradecer a todos la ayuda que me han ofrecido a lo largo de este proyecto. En primer lugar, quiero dar las gracias a mis directores de tesis Dr. Mario David Cordero Morales y Dr. Pedro Bullón Fernández por todo el apoyo y confianza que han depositado en mí. Agradecer a Pedro por haber estado siempre dispuesto a ayudarme en todo lo que he necesitado día a día. Debo agradecer de manera muy especial a mi director y tutor Dr. Mario David Cordero Morales por creer mí, por el interés que siempre le ha mostrado a mis ideas, por su disponibilidad y su gran paciencia. Tengo que agradecerle, no solo el apoyo en el desarrollo de la tesis y en mi formación como investigadora sino también, a su incondicional apoyo tanto en lo laboral como en lo personal. Siempre ha estado ahí y me ha ayudado cuando lo necesitaba y me ha enseñado a desenvolverme en el mundo de la Ciencia. Quiero agradecer a mi grupo de laboratorio por ayudarme en todo momento. A Fabiola por acogerme y enseñarme todo sus conocimientos; a Elizabeth por toda su ayuda y consejos; a Jéssica por su ayuda en el inicio del proyecto, por su alegría y energía, por nuestros almuerzos juntas, por hacerme reír continuamente y por su amistad. Y a mi última compañera y no menos importante, Deborah. Ella ha sido fundamental en esta última etapa, me ha ayudado y apoyado muchísimo, siempre con una sonrisa; muchas gracias por tu amistad. 264 Agradecer a ACTAYS, en especial a Beatriz y Natalia, la confianza depositada en mí y la beca que me ofrecieron para el estudio de estas enfermedades, sin la cual este proyecto no hubiera sido posible. Quiero hacer especial mención al Dr. José Antonio Sánchez Alcázar por abrirme las puertas de su laboratorio y darme la oportunidad de trabajar con él, considerándome una más de su grupo. Y agradecerle el tiempo que dedicó en el inicio de mi andadura en el mundo de la investigación, sin él no habría llegado a donde estoy. También agradecer todo la ayuda y apoyo que encontré en su grupo, siempre dispuestos a facilitarme el trabajo. En primer lugar, agradecerle a Ana todo lo que me enseñó en mi primera etapa. Agradecer a Juanmi toda la ayuda y consejos que me ha ofrecido. Quiero expresar mi más sincero agradecimiento a Marina y a Mónica. Ellas han sido dos pilares muy importantes para mí, siempre dispuestas a ayudarme en cualquier momento y a cualquier hora. Ha sido y será siempre un placer trabajar con ellas porque además de compañeras, las considero muy buenas amigas. Agradecer también su inagotable paciencia y todos los conejos que me han dado. También darle las gracias al Dr. Antonio Pérez Pulido y a su grupo por su ayuda y aclaración de dudas del análisis bioinformático del proyecto. Gracias al Dr. Manuel J. Muñoz y a la Dra. Ana María Brokate Llanos por aceptarme en su laboratorio para la realización de una colaboración y hacer mi estancia muy agradable. Agradezco al Dr. Jon Lane el abrirme las puertas de su laboratorio para la realización de la estancia, por su ayuda desinteresada y por su generosidad para compartir su experiencia y conocimientos. Igualmente, agradecer a su grupo de investigación por facilitarme mi trabajo diario con simpatía. A lo largo de mi trayectoria investigadora he conocido a profesionales muy interesantes y comprometidos con la investigación como son Dr. José M. Álvarez Suárez y Dr. Jordi Muntané Relat. Especialmente agradecer a Jordi por contar conmigo para trabajos de investigación y por su agradable trato.